Rna aptamers and methods for identifying the same

ABSTRACT

RNA aptamers and methods for identifying the same are disclosed. The RNA aptamers selectively bind coagulation factors, E2F family members, Ang1 or Ang2, and therapeutic and other uses for the RNA aptamers are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/963,827 filed Sep. 26, 2001, allowed, which claims priority to U.S.Provisional Application Ser. No. 60/235,654, filed Sep. 26, 2000, hereinincorporated by reference in its entirety.

GRANT STATEMENT

This work was supported by grants R01 HL57606 and R01 CA79983 from theNational Institutions of Health (NIH), and by grant SPS #1805 from theJane Coffin Childs Foundation. Thus, the United States Government hascertain rights in the invention.

TECHNICAL FIELD

The present invention relates generally to compositions and methods foridentifying oligonucleotide sequences that specifically bindbiomolecules, including peptides, hydrophobic molecules, and targetfeatures on cell surfaces, in particular extracellular proteins, and theuse of these sequences to detect and/or isolate the target molecules andthe resulting compositions. The instant invention is exemplified byobtaining compositions, through the use of disclosed methods, thatcomprise oligonucleotide sequences that bind to coagulation factors, E2Ffamily transcription factors, Ang1, Ang2, and fragments or peptidesthereof. Even more particularly, the present invention is directedtowards ribonucleic acid (RNA) aptamers, and to methods of identifyingthem.

TABLE OF ABBREVIATIONS

-   -   I-XII . . . coagulation factors    -   a . . . activated, as in factor VIIa, IXa, Xa, etc.    -   ACT . . . activated clotting time    -   Ang1 . . . Angiopoietin-1    -   Ang1 . . . activated Angiopoietin-1    -   Ang2 . . . Angiopoietin-2    -   Ang2* . . . activated Angiopoietin-2    -   aPTT . . . Activated Partial Thromboplastin Time    -   DNA . . . deoxyribonucleic acid    -   DP . . . diphosphate    -   FB . . . fraction bound    -   GTP . . . guanidine triphosphate    -   K_(d) . . . dissociation constant    -   M7G . . . 5-adenosyl-L-methonine substituted GTP    -   MP . . . monophosphate    -   nM . . . nanomolar    -   PAC . . . phenoxyacetyl    -   pM . . . picomolar    -   PT . . . Prothrombin Time    -   RNA . . . ribonucleic acid    -   SELEX . . . Systematic Evolution of Ligands by EXponential        Enrichment    -   sem . . . standard error for mean    -   TAC . . . t-butoxyacetyl    -   TOG . . . toggle    -   TP . . . triphosphate

BACKGROUND ART

Thrombin belongs to the group of serine proteases and plays a centralpart in the blood coagulation cascade as terminal enzyme. Both theintrinsic and the extrinsic coagulation cascade lead, via a plurality ofamplifying stages, to the production of thrombin from prothrombin.Thrombin-catalyzed cleavage of fibrinogen to fibrin then initiates bloodcoagulation and aggregation of platelets that, in turn, due to thebinding of platelet factor 3 and coagulation factor XIII and a largenumber of highly active mediators, enhance thrombin formation.

To elaborate, the mechanism of blood coagulation normally occurs in acascade of two possible routes. One of the routes, the extrinsic bloodcoagulation, starts with the liberation of thromboplastin and theactivation of factor VII. Activated factor VII (i.e. factor VIIa) inturn activates factor X, followed by an activation of factor V andfactor II (prothrombin). Factor IIa (thrombin) converts fibrinogen intofibrin at the end of the cascade.

The other route, the intrinsic blood coagulation, occurs via anactivation of factor XII by contact with and subsequent activation offactor XI, factor IX and factor X in the presence of calcium and factorVIII, followed by an activation of thrombin (factor II) to activatedthrombin (factor IIa) which triggers coagulation by cleaving fibrinogento fibrin. Thus, activated thrombin (factor IIa) plays a role in bothroutes of the blood coagulation cascade.

Hitherto, there has been an intensive search for anticoagulants that canparticularly be utilized in the treatment of cardiovascular disease,e.g. septic shock, thromboses, embolisms, atherosclerosis and cardiacinfarctions, furthermore in case of blood transfusions or followingsurgery. One method of suppressing the coagulation of blood is thedirect administration of substances that modulate thrombin or othercoagulation factors. The identification of such substances thusrepresents a long-felt and ongoing need in the art.

E2F refers to a family of transcription factors (also referred to hereinas the “E2F family”, which includes but is not limited to E2F-1, E2F-2,E2F-3, E2-F4, E2-F5 and E2-F6), and E2F activity is plays a role in awide variety of proliferative events. E2F activity is controlled as theend result of G₁ cyclin dependent kinase regulatory cascades thatinvolve the Rb family of proteins. See, e.g., Sherr, C. J., Cell,73:1059-1065 (1993); Hunter, T., Cell 75:839-841 (1993); Nevins J. R.,Science, 258:424-429 (1992); Helin, K. and Harlow, E., Trends Cell Biol.3:43-46 (1993); La Thangue, N. B., Trends Biochem. Sci. 19:180-114(1994); Sherr, C. J.; Roberts, J. M., Genes Dev. 9:1149-1143 (1995);Weinberg, R. A. Cell 81:323-330 (1995); Harbour, J. W. and Dean, D. C.,Genes and Development 14:2393-2409 (2000); and Black, A. R. andAzizkhan-Clifford, J., Gene 237:281-302 (1999). Thus, ligands thatselectively bind to an E2F family member would play a role in thecontrol of cell proliferation, which is of central importance to theproper development of a multi-cellular organism, the homeostaticmaintenance of tissues, and the ability of certain cell types to respondappropriately to environmental cues.

Tie2 is an endothelial receptor tyrosine kinase (RTK) that is requiredfor both embryonic vascular development and pathological angiogenesis.Tie2 is unique among RTKs in that it has two ligands with apparentlyopposing actions. Angiopoietin-1 (Ang1) is an activating ligand whileAngiopoietin-2 (Ang2) is thought to be a naturally occurring antagonistfor Tie2. Mice lacking Tie2 or Ang1 die midway through gestation due toabnormalities of vascular morphogenesis characterized by deficientrecruitment of supporting smooth muscle cells and pericytes. Moreover,Ang1 promotes endothelial cell survival and blocks the increases invascular permeability induced by vascular endothelial growth factor(VEGF), supporting a role for Ang1 in the stabilization and maintenanceof the adult vasculature. In contrast, Ang2 is required forVEGF-mediated angiogenesis, and in the absence of endothelial mitogensAng2 may induce vascular regression.

The exact mechanism of action of the Angiopoietins remains to beelucidated. For example, high-dose Ang2 can induce downstream activationof Akt and endothelial cell survival, suggesting that it does not simplyexert a dominant negative effect on Tie2. The need for improvedunderstanding of these ligands' function is particularly important inthe study of tumor angiogenesis, as several studies have now shown thatinhibition of Tie2 with a soluble receptor blocks tumor growth,angiogenesis, and metastasis. However, it is unclear whether theseeffects are due to inhibition of the effects of Ang1 or Ang2, since asoluble receptor would bind both ligands. Specific inhibitors of theseligands have the potential to more precisely modulate Tie2 signaling andserve as valuable therapeutic agents.

Aptamers can comprise single-stranded or double-stranded nucleic acidsthat are capable of binding proteins or other small molecules. Aptamersthat have therapeutic value would most likely bind proteins involved inregulatory cascades. The presence of the aptamer would act as a sink forthe protein factors, preventing the factors from carrying out theirnormal functions. To date, only a few aptamers are known.

It would be desirable to identify novel aptamers that bind to factors inthe coagulation cascade, to an E2F family member, or to an angiogenesisfactor. Indeed, among other applications, such aptamers have utility inthe modulation of coagulation, cell proliferation or angiogenesis, andwould thus meet a long-felt and continuing need in the art.

SUMMARY OF THE INVENTION

An RNA aptamer that selectively binds a coagulation pathway factor, thatselectively binds an E2F family member, or that selectively binds anangiogenesis factor (e.g. Ang1 and/or Ang2) is provided in accordancewith the present invention. Preferably, the binding affinity for the RNAaptamer for the coagulation pathway factor is represented by adissociation constant of about 20 nanomolar (nM) or less. Optionally,the coagulation pathway factor is selected from the group consisting ofthrombin, activated thrombin, Ixa, X, Xa, VII, VIIa and combinationsthereof. Most preferably, the RNA aptamers selectively bind activatedcoagulation factors.

A method of modulating the biological activity of a coagulation pathwayfactor is also provided in accordance with the present invention. Themethod comprises: (a) administering to a warm-blooded vertebrate in needthereof an effective amount of an RNA aptamer that selectively binds acoagulation pathway factor, the RNA aptamer having a dissociationconstant for the coagulation pathway factor of about 20 nM or less; and(b) modulating the biological activity of the coagulation pathway factorin the warm-blooded vertebrate through the administering of the RNAaptamer in step (a).

A method of treating cardiovascular disease in a warm-blooded vertebrateis also provided in accordance with the present invention. The methodcomprises administering an effective amount of an RNA aptamer thatselectively binds a coagulation pathway factor, the RNA aptamer having adissociation constant for the coagulation pathway factor of about 20 nMor less, to a vertebrate subject suffering from cardiovascular disease,whereby cardiovascular disease in the vertebrate subject is treated.

A method of modulating E2F activity in a warm-blooded vertebrate inwhich said modulation is desired is also provided. The method comprises:(a) administering to the warm-blooded vertebrate an effective amount ofan RNA aptamer that selectively binds an E2F family member, the RNAaptamer having a dissociation constant for the E2F family member ofabout 20 nM or less; and (b) modulating E2F in the warm-bloodedvertebrate through the administering of the RNA aptamer of step (a).

A method of modulating Ang1 or Ang2 activity in a warm-bloodedvertebrate in which said modulation is desired is also provided. Themethod comprises: (a) administering to the warm-blooded vertebrate aneffective amount of an RNA aptamer that selectively binds Ang1 or Ang2,the RNA aptamer having a dissociation constant for Ang1 or Ang2 of about20 nM or less; and (b) modulating Ang1 or Ang2 in the warm-bloodedvertebrate through the administering of the RNA aptamer of step (a).

A method of identifying a ligand to a target from a candidate mixture ofpotential ligands is also provided in accordance with the presentinvention. Products, i.e., ligands, produced or identified via themethod are also provided in accordance with the present invention.

In a preferred embodiment the method comprises: (a) preparing acandidate mixture of potential ligands; (b) contacting the candidatemixture with a target in a lower stringency buffer, wherein ligandshaving increased affinity to the target relative to the candidatemixture bind to the target; (c) removing unbound candidate mixture; and(d) collecting the ligands that are bound to the target to produce afirst collected ligand mixture. More preferably, the method furthercomprises: (e) contacting the first collected ligand mixture with thetarget in a higher stringency buffer, wherein ligands having increasedaffinity to the target relative to the first collected ligand mixturebind to the target; (f) removing unbound ligands; and (g) collecting theligands that are bound to the target to produce a second collectedligand mixture to thereby identify ligands to the target. Even morepreferably, ligands in the first or second collected ligand mixture areenriched or expanded by any suitable technique, e.g. amplification,prior to contacting the first collected ligand mixture with the targetin the higher stringency buffer, after collecting the ligands that boundthe target in the higher stringency buffer, or both. Optionally, thecontacting and expanding or enriching steps are repeated as necessary toproduce a desired ligand. Thus, it is possible that the second collectedligand mixture can comprise a single ligand.

Another embodiment of a method of identifying a ligand to a target froma candidate mixture of potential ligands is provided in accordance withthe present invention. Products, i.e., ligands, produced or identifiedvia the method are also provided in accordance with the presentinvention.

The method preferably comprises: (a) providing a target selected from afirst species of organism; (b) preparing a candidate mixture ofpotential ligands; (c) contacting the candidate mixture with the target,wherein ligands having increased affinity to the target from the firstspecies of organism relative to the candidate mixture bind to the targetfrom the first species of organism; (d) removing unbound candidatemixture; (e) collecting the ligands that are bound to the target fromthe first species of organism to produce a first collected ligandmixture for the target; (f) contacting the first collected ligandmixture with a target from a second species of organism, the target fromthe second species having at least a portion thereof that issubstantially homologous to the same portion in the target from thefirst species, wherein ligands having increased affinity to the targetfrom the second species relative to the first collected ligand mixturebind to the target; (g) removing unbound first collected ligand mixture;and (h) collecting the ligands that are bound to the target from thesecond species of organism to form a second collected ligand mixturethereby identify ligands to the target.

Preferably, ligands in the first or second collected ligand mixture areenriched or expanded by any suitable technique, e.g. amplification,prior to contacting the first collected ligand mixture with the targetfrom the second species of organism, after collecting the ligands thatbound the target from the second species of organism, or both.Optionally, the contacting and expanding or enriching steps are repeatedas necessary to produce a desired ligand. Thus, it is possible that thesecond collected ligand mixture can comprise a single ligand.

It is therefore an object of the present invention to provide novel RNAaptamers that selectively bind coagulation factors, an E2F familymember, Ang1 or Ang2. The object is achieved in whole or in part by thepresent invention.

An object of the invention having been stated hereinabove, other objectswill become evident as the description proceeds, when taken inconnection with the accompanying Figures and Laboratory Examples as bestdescribed herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts sequences (SEQ ID NOs:1-12) of representative RNAaptamers of the present invention that bind to and inhibit the activityof coagulation factor IXa.

FIG. 1B depicts sequences (SEQ ID NOs:13-22) of representative RNAaptamers of the present invention that bind to and inhibit the activityof coagulation factor IXa.

FIG. 2A is a plot of data points showing that RNA 9.3 (SEQ ID NO:3)inhibits the FIXa/FVIIIa catalyzed activation of FX, therebydemonstrating that RNA 9.3 (SEQ ID NO:3) inhibits the activity ofcoagulation factor IXa. Factor IXa (0.5 nM) was equilibrated with no RNA(▴), 10 nM control RNA (▪), or 10 nM RNA 9.3 (SEQ ID NO:3) (), the FXactivation reaction was initiated by the addition of FVIIIa (1 nM),PC/PS vesicles (100 μM) and FX (200 nM), and the amount of FXa formedover time at 37° C. was measured.

FIG. 2B is a bar graph showing that RNA 9.3 (SEQ ID NO:3) prolongs theclotting time of human plasma, thereby demonstrating RNA 9.3 (SEQ IDNO:3) inhibits the activity of coagulation factor IXa. The clotting timeof normal human plasma was measured in an aPTT assay in the absence ofRNA (striped bar), or in the presence of increasing concentrations ofcontrol RNA (solid bar) or RNA 9.3 (SEQ ID NO:3) (open bar). Theclotting time is expressed as the mean ±sem for duplicate measurements.

FIGS. 3A-3C show that aptamer 9.3t (SEQ ID NO:70) inhibits FIXa activityin pigs.

FIG. 3A is a plot showing in vitro anticoagulant activity of aptamer9.3° in porcine plasma. ▪, 9.3^(t) APTT; □, 9.3^(t) PT; 9.3^(tm) APTT;O, 9.3^(tm) PT. Data is presented as the mean ±sem.

FIG. 3B is a line graph showing in vivo anticoagulant activity ofaptamer 9.3t in pigs following IV bolus injection, Activated ClottingTime (ACT) assays. ▪, 1.0 mg/kg 9.3^(t.); □, 0.5 mg/kg 9.3^(t.); , 1.0mg/kg 9.3^(tm); O, Vehicle. Data is presented as the mean ±sem. The ACTincrease is the ratio of the (pre-injection ACT/post injection ACT) foreach time point, 1.0=no change (time 0=pre-injection).

FIG. 3C is a plot showing in vivo anticoagulant activity of aptamer 9.3tin pigs following IV bolus injection, Prothrombin Time (PT) andactivated Partial Thromboplastin Time (aPTT) assays. ▪, 1.0 mg/kg9.3^(t.); □, 0.5 mg kg 9.3^(t.); , 1.0 mg/kg 9.3^(tM); O, Vehicle. Datais presented as the mean ±sem. The increase in the PT or APTT is theratio of the (pre-injection clot time/post injection clot time) for eachtime point, 1.0=no change (time 0=pre-injection).

FIG. 4A is a line graph showing in vitro inhibitory activity ofcholesterol modified aptamer 9.3^(t) (SEQ ID NO:70—cholesterol modifiedform referred to herein as 9.3^(t-C)). Cholesterol addition has modesteffect on the affinity of aptamer 9.3^(t-C) for FIXa based upon acompetition binding assay to measure affinity of 9.3^(t-C) for FIXa.Competitors: ▪, 9.3^(t.); O, 9.3^(t-C); Δ, 93^(tM).

FIG. 4B is a plot depicting in vitro anticoagulant activity of aptamer9.3^(t-C) in human plasma. ▪, 9.3^(t-C) APTT; □, 9.3^(t-C) PT; ,93^(tM-C) APTT, O, 9.3^(tM-C) PT.

FIG. 4C is a plot depicting in vitro anticoagulant activity aptamer9.3^(t-C) in pig plasma. ▪, 9.3^(t-C) APTT; □, 9.3^(t-C) PT; □,9.3^(tM-C) APTT; O, 9.3^(tM-c) PT.

FIGS. 5A-5C depict in vivo anticoagulant activity of aptamer 9.3^(t-C).

FIG. 5A is a line graph showing in vivo anticoagulant of aptamer9.3^(t-C) in pigs following IV bolus injection, ACT assays. ▪, 0.5 mg/kg9.3^(t-C); □, 0.5 mg/kg 9.3^(tM-C); dotted line is 9.3^(t) ACT data at0.5 mg/ml from FIG. 3B. Data is presented is the average of duplicatemeasurements at each time point.

FIG. 5B is a plot showing in vivo plasma concentration of 9.3^(t-C)(solid circle) versus 9.3^(t) (solid square) over time following bolusIV injection. Concentrations calculated by interpolation from in vitrodose response curves of APTT assays for each aptamer.

FIG. 5C is a plot showing in vivo anticoagulant of aptamer 9.3^(t-C) inpigs following IV bolus injection, APTT and PT assays. 0.5 mg/kg9.3^(t-C); 0.5 mg/kg 9.3^(tM-C); *0.5 mg/kg 9.3^(t) from FIG. 3C; □,9.3^(t-C) APTT; □, 9.3^(t-C) PT; , 9.3^(tM-C) APTT; O, 9.3^(tM-C) PT.Data is presented is the average of duplicate measurements at each timepoint.

FIG. 6A depicts sequences (SEQ ID NO: 23-30 and 73) of RNA aptamers thatbind to and inhibit the activity of coagulation factor X and/or itsactivated form factor FXa.

FIG. 6B depicts sequence (SEQ ID NOS: 31-38) of RNA aptamers that bindto and inhibit the activity of coagulation factor X and/or its activatedform factor FXa.

FIG. 7A is a plot of data points depicting that RNA 10.14 (SEQ ID NO:73)inhibits the FXa/FVa catalyzed activation of prothrombin, therebydemonstrating that RNA 10.14 inhibits the activity of coagulation factorXa. Factor Xa (0.5 nM) was equilibrated with no RNA (▴), 100 nM controlRNA (▪), or 100 nM RNA 10.14 (, SEQ ID NO:73). The prothrombinactivation reaction was initiated by the addition of FVa (1 nM), PC/PSvesicles (100 μM) and prothrombin (200 nM), and the amount of thrombinformed over time at 37 C was measured.

FIG. 7B is a bar graph depicting clotting time, thereby depicting thatRNA 10.14 inhibits the activity of coagulation factor Xa The clottingtime of normal human plasma was measured in a PT assay in the absence ofRNA (striped bar), or in the presence of increasing concentrations ofcontrol RNA (solid bar) or RNA 10.14 (open bar). The clotting time isexpressed as the mean ±sem for duplicate measurements.

FIG. 8 is a PHOSPHORIMAGER® (Molecular Dynamics of Sunnyvale, Calif.)scan depicting initial condition matrix to coagulation factor VIIa.Radiolabeled 2′fluoropyrimidine-modified library RNA was incubated withvarying concentrations of coagulation factor VIIa in 9 differentbuffers, and target-bound versus free RNA was determined using thedouble-filter nitrocellulose filter binding assay (Wong and Lohman,1993, Proc. Natl. Acad. Sci. USA 90, 5428-5432).

FIG. 9 is a PHOSPHORIMAGER® (Molecular Dynamics of Sunnyvale, Calif.)scan of an initial condition matrix to coagulation factor IXa.Radiolabeled 2′fluoropyrimidine-modified library RNA was incubated withvarying concentrations of coagulation factor IXa in 6 different buffers,and target-bound versus free RNA was determined using the double-filternitrocellulose filter binding assay.

FIG. 10 depicts sequences (SEQ ID NOS:39-47) of RNA aptamers that bindto and inhibit the activation of coagulation factor VIIa.

FIG. 11 is a table depicting full-length thrombin aptamer sequences (SEQID NOs:50-56) produced by the toggle SELEX method of the presentinvention. The noted sequences are proceeded 5′ by the followingsequence: GGGAGAGAGGAAGAGGGAUGGG (SEQ ID NO: 48); and the notedsequences are followed 3′ by the following sequence:CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO: 49).

FIG. 12 is a bar graph depicting human platelet activation by 1 nM humanthrombin, in the presence of 30 nM TOG 25 RNA aptamer (SEQ ID NO: 55) or30 nM nitrocellulose-control aptamer.

FIG. 13 is a bar graph depicting pig platelet activation by 2 nM pigthrombin, in the presence of 25 nM TOG 25 aptamer (SEQ ID NO: 55) or 25nM nitrocellulose binding control aptamer.

FIG. 14 is a line graph depicting activated partial thromboplastin timeassays that were performed on pig (solid diamonds) and human (solidsquares) plasma in the presence of increasing concentrations of TOG 30aptamer (SEQ ID NO: 56). Control aptamer had no effect on APTT at theseconcentrations.

FIG. 15 is a schematic of a proposed structure of a 25-MER truncate ofTOG 25 (TOG 25 short, SEQ ID NO: 57). The “wild-type” truncate bindshuman thrombin with a K_(d) of about 1 nM. The binding affinity ofvarious mutants designed to disrupt the stem (no stem, SEQ ID NO: 58),BULGE (BULGE Us, SEQ ID NO: 59), and loop sequence (loop U1, SEQ ID NO:60, and loop U2, SEQ ID NO: 61) are also shown.

FIG. 16 is a line graph depicting activation of human platelets by 1 nMhuman thrombin in the presence of increasing concentrations of fulllength TOG 25 (TOGFL, SEQ ID NO:55, solid diamonds), truncated TOG 25(TOG 25 short, SEQ ID NO: 57, solid squares), or a non-binding mutanttruncate (BULGE Us, SEQ ID NO: 59, solid triAng1 es).

FIG. 17 depicts sequences (SEQ ID NOs:64-69) of RNA aptamers that bindto and inhibit the activity of E2F. The aptamers were produced after 10rounds of the SELEX method and each primer includes an oligonucleotidesequence (SEQ ID NO:62) at the 5′ end and at the 3′ end (SEQ ID NO:63)as noted. In FIG. 17, and in other parts of the application, if a T isobserved in an RNA sequence it should be construed as a U, in that theinclusion of the T is an unintended artifact of a sequencing approach.

FIG. 18 is a table depicting the alignment of a family of FIX/FIXAaptamers of the present invention and depicting a proposed secondarystructure of truncated form 9.3^(t) (SEQ ID NO:70) of aptamer 9-3 (SEQID NO: 3) of FIG. 1. In FIG. 18, S1 equals stem 1, L1 equals loop 1, S2equals stem 2, L2 equals loop 2, and L3 equals loop 3.

FIG. 19A is a line graph depicting that RNA 16.3 (SEQ ID NO: 41)inhibits the FVIIa/TF catalyzed activation of FX. Factors VIIa and Xwere equilibrated with no RNA (□), 1 μM RNA 16.3 m4 (▪), or 1 μM RNA16.3 (▴). The FX activation reaction was initiated by the addition oflipidated tissue factor, and the amount of FXa formed over time at 25°C. was measured. The amount of FXa formed over time is expressed as themean ±sem for three more experiments.

FIG. 19B is a bar graph that depicts that RNA 16.3 (SEQ ID NO: 41)prolongs the tissue factor induced clotting time of human plasma. Theclotting time of normal human plasma was measured in a PT assay in theabsence of RNA (striped bar), wherein the presence of varyingconcentrations of 16.3 m4 (solid bar) or RNA 16.3 (open bar). Theclotting time is expressed as the mean ±SEM for duplicate experiments.

FIG. 20 depicts the secondary structure of coagulation factor IXa RNAaptamer 9.3^(t) (SEQ ID NO:70). The A-G substitutions that are used tocreate the mutant control aptamer 9.3^(tM) (K_(D) for factor IXa>>5 μM)are also shown. S1 equals stem 1, L1 equals loop 1, S2 equals stem 2, L2equals loop 2, and L3 equals loop 3.

FIG. 21 depicts the secondary structure of the minimal form of thecoagulation FXa aptamer 11.F7^(t) (SEQ ID NO: 148) that retains fullbinding and inhibitory activity. Secondary structure was predicted by acombination of comparative sequence analysis and RNA folding algorithms.

FIG. 22A is a histogram showing prothrombin time (PT) clotting assays inhuman plasma that demonstrate the anticoagulant activity of 11.7Ft (SEQID NO: 148). Dashed lines in histogram represents baseline clottingtimes in the presence of non-FXa binding control aptamers of similarlength.

FIG. 22B is a histogram showing activated partial thromboplastin time(APTT) clotting assays in human plasma that demonstrate theanticoagulant activity of 11.F7^(t) (SEQ ID NO: 148). Dashed lines inhistogram represents baseline clotting times in the presence of non-FXabinding control aptamers of similar length.

FIGS. 23A and 23B depict binding and inhibitory activity of FVIIaaptamers 10.15 (SEQ ID NO: 75, solid squares) and 11.12 (SEQ ID NO:88,solid diamonds).

FIG. 23A is a line graph showing homologous competition binding assaysthat demonstrate high affinity binding of both aptamers to FVIIa.

FIG. 23B is a plot showing prothrombin time (PT) clotting assays inhuman plasma that demonstrate the anticoagulant activity of bothaptamers. PT's normalized to a baseline measurements (baseline=˜12 sec.)in absence of aptamer, therefore a value of i=no effect.

FIG. 24 is a line graph wherein direct binding (corrected fraction bound(FB)) of ANG9-4 RNA aptamer (SEQ ID NO:151) is plotted as a function ofprotein concentration. ANG9-4 binds Ang1 (solid circles) with a K_(d) of˜10 nM and binds the related antagonist Ang2 with a K_(d) of >1 μM(>100-fold specificity)(solid triangles).

FIG. 25 depicts a Western blot and table that were prepared to determinewhether ANG9-4 binding inhibited Ang1 activity. 293 cells expressinghuman Tie2 were incubated with 13 nM Ang1* with or without a molarexcess of 9-4 or control aptamer. ANG9-4 completely abrogated Tie2autophosphorylation as detected by Western blotting with an antibodyspecific for phospho-Tie2 (pTie2).

FIG. 26 is a histogram and table showing effect on apoptosis of Ang1aptamers. Cultured human endothelial cells were serum-starved andtreated with TNFα (50 ng/ml) for 3 hours while being incubated with 3.5nM Ang1* and either ANG9-4 or control aptamer. Apoptosis (DNAfragmentation) was measured by Cell Death Detection ELISA kit (RocheMolecular Biochemicals). ANG9-4, but not control aptamer, increasedapoptosis in a dose-dependent manner. Neither 9-4 nor control RNAincreased apoptosis in un-starved, untreated cells.

FIG. 27 is a line graph wherein direct binding (corrected fraction bound(FB)) of ANG11-1 RNA is plotted as a function of protein concentration.ANG11-1 binds Ang2 (solid triAng1 es) with a K_(d) of <10 nM and bindsAng1* with a K_(d) of ˜1 μM (>100-fold specificity)(solid circles).

FIG. 28 is a histogram and table showing effect on apoptosis of Ang2aptamers. Cultured human endothelial cells were serum-starved andtreated with TNFα (50 ng/ml) for 3 hours while being incubated with 15nM Ang2 and either ANG11-1 or control aptamer. Apoptosis (DNAfragmentation) was measured by Cell Death Detection ELISA kit (RocheMolecular Biochemicals). ANG11-1, but not control aptamer, increasedapoptosis to a levels above those seen in the absence of exogenous Ang2,suggesting inhibition of both exogenous and endogenous (autocrine) Ang2,which is known to be released by endothelial cells. Neither ANG11-1 norcontrol RNA increased apoptosis in non-irradiated cells.

FIG. 29 is a line graph wherein the ability of ANG11-1 and a41-nucleotide truncate (ANG11-1.41) to compete for binding to ANG2(normalized to binding in the absence of competitor) is plotted as afunction of competitor concentration. ANG11-1.41 (solid squares)competes for ANG2 binding with an affinity (K_(d)−5 nM) only slightlyworse that of the full-length RNA aptamer (˜1 nM)(solid diamonds).

FIG. 30 is a secondary structure schematic of ANG11-1.41, prepared viaapplication of sequence alignment and RNA-folding algorithms to thepredominant sequence family suggests a common stem-looped structure,which incorporated the conserved sequence motif. Scrambling the6-nucleotide consensus loop sequence (CAGCUC>ACUCCG) disrupts theability of the truncate to bind Ang2 (K_(d)>1000 nM), as does mutationof the sequence comprising the terminal stem ((K_(d)−1000 nM).

FIG. 31 is a schematic depicting the Toggle SELEX method of the presentinvention. Aptamers that bind both human and porcine thrombin wereselected by “toggling” the protein target between human and porcinethrombin during alternating rounds of selection.

FIGS. 32A-32F depict RNA pool binding. The fraction of RNA bound,corrected for nonspecific binding to nitrocellulose (Corrected FB), isplotted as a function of thrombin concentration. The binding affinitiesof RNA pools from the toggle selection (, circle), human thrombinselection (▪, square), and porcine thrombin selection (♦, diamond) forhuman thrombin protein (FIGS. 32A, 32C, 32E) and porcine thrombinprotein (FIGS. 32B, 32D, 32F) were compared after round 3 (FIGS. 32A,32B), round 11 (FIGS. 32C, 32D), and round 13 (FIGS. 32E, 32F).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,biochemistry, protein chemistry, and recombinant DNA technology, whichare within the skill of the art. Such techniques are explained fully inthe literature. See, e.g., Oligonucleotide Synthesis (M. J. Gait ed.1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.1984); Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, Second Edition (1989); and the series Methods in Enzymology (S.Colowick and N. Kaplan eds., Academic Press, Inc.).

Disclosed herein are RNA aptamer molecules that modulate, andpreferably, that inhibit the activities of Factor VII, IX, X andthrombin. Also disclosed herein are E2F RNA aptamers that have beenshown to bind E2F family members including E2F-3 to thereby modulate thebiological activity of the E2F. Also disclosed herein are RNA aptamersthat have been shown to bind Ang1 and Ang2 to thereby modulate thebiological activity of Ang1 and Ang2. Optionally, the aptamers areidentified through a method known in the art as Systematic Evolution ofLigands by EXponential Enrichment, SELEX; preferably, the aptamers areidentified by a modified or toggle SELEX methods as disclosed herein.

The RNA aptamers of the present invention preferably comprise2′fluoro-pyrimidines to enhance resistance to nuclease degradation. Theaffinities of the present inventive RNA ligands for the various factorspreferably range from K_(d)s of about 100 pM to about 10 nM. Inaddition, these RNA ligands can act as competitive inhibitors and blockfactors VIIa, Ixa, Xa, and thrombin activity in enzymatic assays.Moreover, these RNA ligands can act as potent anticoagulants andsignificantly delay the clotting time of normal human plasma or theactivation of platelets in response to thrombin. The RNA aptamer ligandsfor E2F are useful for inhibiting cell proliferation in a number ofclinical settings including but not restricted to intimal hyperplasiafollowing bypass graft surgery. The RNA aptamers that have been shown tobind Ang1 and Ang2 to thereby modulate the biological activity of Ang1and Ang2 can be used in the modulation of angiogenesis.

Also disclosed herein are in vitro selection techniques andcombinatorial chemistry methods for identifying and isolating, amongother ligands, RNA aptamer molecules that bind to human coagulationfactor VII, VIIa, IX, IXa, X, Xa, thrombin and activated thrombin, E2Ftranscription factors, and Ang1 and Ang2 with high affinity andspecificity.

I. DEFINITIONS

The following terms are believed to have well-recognized meanings in theart. However, the following definitions are set forth to facilitateexplanation of the invention.

As used herein, a “target” or “target molecule” refers to a biomoleculethat could be the focus of a therapeutic drug strategy or diagnosticassay, including, without limitation, proteins or portions thereof,enzymes, peptides, enzyme inhibitors, hormones, carbohydrates,glycoproteins, lipids, phospholipids, nucleic acids, and generally, anybiomolecule capable of turning a biochemical pathway on or off ormodulating it, or which is involved in a predictable biologicalresponse. Targets can be free in solution, like thrombin, or associatedwith cells or viruses, as in receptors or envelope proteins. Any ligandthat is of sufficient size to be specifically recognized by anoligonucleotide sequence can be used as the target. Thus, glycoproteins,proteins, carbohydrates, membrane structures, receptors, organelles, andthe like can be used as the complexation targets.

A wide variety of materials can serve as targets. These materialsinclude intracellular, extracellular, and cell surface proteins,peptides, glycoproteins, carbohydrates, including glycosaminoglycans,lipids, including glycolipids and certain oligonucleotides.

The term “ligand” as used herein refers to a molecule or other chemicalentity having a capacity for binding to a target. A ligand can comprisea peptide, an oligomer, a nucleic acid (e.g., an aptamer), a smallmolecule (e.g., a chemical compound), an antibody or fragment thereof,nucleic acid-protein fusion, and/or any other affinity agent. Thus, aligand can come from any source, including libraries, particularlycombinatorial libraries, such as the aptamer libraries disclosed hereinbelow, phage display libraries, or any other library as would beapparent to one of ordinary skill in the art after review of thedisclosure of the present invention presented herein.

The term “RNA analog” is meant to refer to a polymeric molecule, whichin addition to containing ribonucleosides as its units, also contains atleast one of the following: 2′-deoxy, 2′-halo (including 2′-fluoro),2′-amino (preferably not substituted or mono- or disubstituted),2′-mono-, di- or tri-halomethyl, 2′-O-alkyl, 2′-O-halo-substitutedalkyl, 2′-alkyl, azido, phosphorothioate, sulfhydryl, methylphosphonate,fluorescein, rhodamine, pyrene, biotin, xanthine, hypoxanthine,2,6-diamino purine, 2-hydroxy-6-mercaptopurine and pyrimidine basessubstituted at the 6-position with sulfur or 5 position with halo orC₁₋₅ alkyl groups, abasic linkers, 3′-deoxy-adenosine as well as otheravailable “chain terminator” or “non-extendible” analogs (at the 3′-endof the RNA), or labels such as ³²P, ³³P and the like. All of theforegoing can be incorporated into an RNA using the standard synthesistechniques disclosed herein.

The terms “binding activity” and “binding affinity” are meant to referto the tendency of a ligand molecule to bind or not to bind to a target.The energetics of said interactions are significant in “bindingactivity” and “binding affinity” because they define the necessaryconcentrations of interacting partners, the rates at which thesepartners are capable of associating, and the relative concentrations ofbound and free molecules in a solution. The energetics are characterizedherein through, among other ways, the determination of a dissociationconstant, K_(d). Preferably, the K_(d) is established using adouble-filter nitrocellulose filter binding assay such as that disclosedby Wong and Lohman, 1993, Proc. Natl. Acad. Sci. USA 90, 5428-5432.Every more preferably, an aptamer of the present invention having apreferred K_(d) value is further evaluated in an assay for effects onthe target. For example, a K_(i) value as described herein below can bedetermined for the aptamer and the target.

As used herein, “specifically binding oligonucleotides”, “nucleic acidligands” or “aptamers” refer to oligonucleotides having specific bindingregions that are capable of forming complexes with an intended targetmolecule in an environment herein other substances in the sameenvironment are not complexed to the oligonucleotide. The specificity ofthe binding is defined in terms of the comparative dissociationconstants (K_(d)) of the aptamer for target as compared to thedissociation constant with respect to the aptamer and other materials inthe environment or unrelated molecules in general. Typically, the K_(d)for the aptamer with respect to the target will be 2-fold, preferably5-fold, more preferably 10-fold less than K_(d) with respect to targetand the unrelated material or accompanying material in the environment.Even more preferably the K_(d) will be 50-fold less, more preferably100-fold less, and more preferably 200-fold less.

The binding affinity of the aptamers herein with respect to targets andother molecules is defined in terms of K_(d). The value of thisdissociation constant can be determined directly by well-known methods,and can be computed even for complex mixtures by methods such as those,for example, set forth in Caceci, M., et al., Byte (1984) 9:340-362. Ithas been observed, however, that for some small oligonucleotides, directdetermination of K_(d) is difficult, and can lead to misleadingly highresults. Under these circumstances, a competitive binding assay for thetarget molecule or other candidate substance can be conducted withrespect to substances known to bind the target or candidate. The valueof the concentration at which 50% inhibition occurs (K_(i)) is, underideal conditions, equivalent to K_(d). However, in no event will aK_(i); be less than K_(d). Thus, determination of K_(i) in thealternative, sets a maximal value for the value of K_(d). Under thosecircumstances where technical difficulties preclude accurate measurementof K_(d), measurement of K_(i) can conveniently be substituted toprovide an upper limit for K_(d). A K_(i) value can also be used toconfirm that an aptamer of the present binds a target.

As specificity is defined in terms of K_(d) as set forth above, incertain embodiments of the present invention it is preferred thatexcluded from the categories of unrelated materials and materialsaccompanying the target in the target's environment are those materialswhich are sufficiently related to the target to be immunologicallycrossreactive therewith. By “immunologically crossreactive” is meantthat antibodies raised with respect to the target crossreact understandard assay conditions with the candidate material. Generally, forantibodies to crossreact in standard assays, the binding affinities ofthe antibodies for crossreactive materials as compared to targets shouldbe in the range of 5-fold to 100-fold, generally about 10-fold.

In general, aptamers preferably comprise about 10 to about 100nucleotides, preferably about 15 to about 40 nucleotides, morepreferably about 20 to about 40 nucleotides, in that oligonucleotides ofa length that falls within these ranges are readily prepared byconventional techniques. Optionally, aptamers can further comprise aminimum of approximately 6 nucleotides, preferably 10, and morepreferably 14 or 15 nucleotides, that are necessary to effect specificbinding. The only apparent limitations on the binding specificity of thetarget/oligonucleotide couples of the invention concern sufficientsequence to be distinctive in the binding oligonucleotide and sufficientbinding capacity of the target substance to obtain the necessaryinteraction. Aptamers of binding regions containing sequences shorterthan 10, e.g., 6-mers, are feasible if the appropriate interaction canbe obtained in the context of the environment in which the target isplaced. Thus, if there is little interference by other materials, lessspecificity and less strength of binding can be required.

As used herein, “aptamer” refers in general to either an oligonucleotideof a single defined sequence or a mixture of said oligonucleotides,wherein the mixture retains the properties of binding specifically tothe target molecule. Thus, as used herein “aptamer” denotes bothsingular and plural sequences of oligonucleotides, as definedhereinabove. The term “aptamer” is meant to refer to a single- ordouble-stranded nucleic acid which is capable of binding to a protein orother molecule, and thereby disturbing the protein's or other molecule'sfunction.

Structurally, the aptamers of the invention are specifically bindingoligonucleotides, wherein “oligonucleotide” is as defined herein. As setforth herein, oligonucleotides include not only those with conventionalbases, sugar residues and internucleotide linkages, but also those thatcontain modifications of any or all of these three moieties.

“Single-stranded” oligonucleotides, as the term is used herein, refersto those oligonucleotides that contain a single covalently linked seriesof nucleotide residues.

“Oligomers” or “oligonucleotides” include RNA or DNA sequences of morethan one nucleotide in either single chain or duplex form andspecifically includes short sequences such as dimers and trimers, ineither single chain or duplex form, which can be intermediates in theproduction of the specifically binding oligonucleotides. “Modified”forms used in candidate pools contain at least one non-native residue.

“Oligonucleotide” or “oligomer” is generic to polydeoxyribonucleotides(containing 2′-deoxy-D-ribose or modified forms thereof), i.e., DNA, topolyribonucleotides (containing D-ribose or modified forms thereof),i.e., RNA, and to any other type of polynucleotide which is anN-glycoside or C-glycoside of a purine or pyrimidine base, or modifiedpurine or pyrimidine base or abasic nucleotides.

An “RNA aptamer” is an aptamer comprising ribonucleoside units. “RNAaptamer” is also meant to encompass RNA analogs as defined herein above.

The term “coagulation factor aptamer” is meant to refer to a single- ordouble-stranded nucleic acid that binds a coagulation factor andmodulates its function. The term “coagulation factor” is meant to referto a factor that acts in either or both of the intrinsic and theextrinsic coagulation cascade.

When a number of individual, distinct aptamer sequences for a singletarget molecule have been obtained and sequenced as described above, thesequences can be examined for “consensus sequences”. As used herein,“consensus sequence” refers to a nucleotide sequence or region (whichmight or might not be made up of contiguous nucleotides) that is foundin one or more regions of at least two aptamers, the presence of whichcan be correlated with aptamer-to-target-binding or with aptamerstructure.

A consensus sequence can be as short as three nucleotides long. It alsocan be made up of one or more noncontiguous sequences with nucleotidesequences or polymers of hundreds of bases long interspersed between theconsensus sequences. Consensus sequences can be identified by sequencecomparisons between individual aptamer species, which comparisons can beaided by computer programs and other tools for modeling secondary andtertiary structure from sequence information. Generally, the consensussequence will contain at least about 3 to 20 nucleotides, more commonlyfrom 6 to 10 nucleotides.

As used herein “consensus sequence” means that certain positions, notnecessarily contiguous, of an oligonucleotide are specified. Byspecified it is meant that the composition of the position is other thancompletely random. Not all oligonucleotides in a mixture can have thesame nucleotide at such position; for example, the consensus sequencecan contain a known ratio of particular nucleotides. For example, aconsensus sequence might consist of a series of four positions whereinthe first position in all members of the mixture is A, the secondposition is 25% A, 35% T and 40% C, the third position is T in alloligonucleotides, and the fourth position is G in 50% of theoligonucleotides and C in 50% of the oligonucleotides.

The terms “cardiovascular disease” and “cardiovascular diseases” aremeant to refer to any cardiovascular disease as would be understood byone of ordinary skill in the art. Examples of particularly contemplatedcardiovascular diseases include, but are not limited to,atherosclerosis, thrombophilia, embolisms, cardiac infarction (e.g.,myocardial infarction), thromboses, angina, stroke, septic shock,hypertension, hyper-cholesterolemia, restenosis and diabetes. Moreparticularly, the terms “cardiovascular disease” and “cardiovasculardiseases” are meant to refer to cardiovascular diseases in whichthrombosis plays a causative, aggravating and/or indicating role.

The phrase “treating cardiovascular disease” is meant to refer to thetreatment of cardiovascular disease at any stage of progression. Thus,treatment of early onset cardiovascular disease as well as treatment ofadvanced cardiovascular disease falls within the scope of the phrase“treating cardiovascular disease”. The phrase “treating cardiovasculardisease” is also meant to refer to a therapeutic method directed towardinhibiting the aggravation of cardiovascular disease by modulatingcoagulation.

The term “truncate” refers to an aptamer that has been truncated bydeletion of nucleotides but still possesses a desired or even improvedbinding characteristic. Truncates can vary in length in accordance withthe length of the starting aptamer and as defined above for the term“aptamer”. Truncations in the truncate can occur in fixed or variableregions, or both fixed and variable regions, of the starting aptamer.

The term “about”, as used herein when referring to a measurable valuesuch as an amount of weight, time, dose, etc. is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified amount, as suchvariations are appropriate to perform the disclosed method.

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

II. RNA APTAMERS

An RNA aptamer that selectively binds a coagulation pathway factor, thatselectively binds an E2F family member, or that that selectively bindsAng1 or Ang2 is provided in accordance with the present invention.Preferably, the binding affinity for the RNA aptamer for the coagulationpathway factor is represented by dissociation constant of about 20nanomolar (nM) or less, more preferably about 10 nanomolar (nM) or less.Optionally, the coagulation pathway factor is selected from the groupincluding but not limited to prothrombin, thrombin, IX, IXa, X, Xa, VII,VIIa, V, Va, VIII, VIIIa, tissue factor, XI, XIa and combinationsthereof. Most preferably, the RNA aptamers bind activated coagulationfactors.

Thus, improved nucleic acid ligands to coagulation factors, to E2Ffamily members, and to Ang1 or Ang2 are disclosed and claimed herein.This invention includes the specific nucleic acid ligands identifiedherein. The scope of the ligands covered by the invention extends to allligands of a coagulation factor, to E2F family members, and to Ang1 orAng2 identified according to the procedures described herein. Morespecifically, this invention includes nucleic acid sequences that aresubstantially homologous to and that have substantially the same abilityto bind coagulation factors, E2F family members, or Ang1 or Ang2, underphysiological conditions, as the nucleic acid ligands identified herein.By substantially homologous, it is meant, a degree of homology in excessof 70%, most preferably in excess of 80%. Alignment techniques aredisclosed herein below. Substantially homologous also includes base pairflips in those areas of the nucleic acid ligands that include basepairing regions. Substantially the same ability to bind a coagulationfactor, an E2F family member, or Ang1 or Ang2 means that the affinity iswithin two orders of magnitude of the affinity of the nucleic acidligands described herein. It is well within the skill of those ofordinary skill in the art to determine whether a given sequence issubstantially homologous to and has substantially the same ability tobind a coagulation factor, an E2F family member, or Ang1 or Ang2 as thesequences identified herein.

II.A. Coagulation Factor IX Aptamers

In one embodiment, an RNA aptamer of the present invention selectivelybinds an activated coagulation factor IXa (FIXa) or inactive formthereof, i.e. (Factor IX). Preferably, the dissociation constant rangesfrom about 100 pM to about 10 nM. More preferably, the dissociationconstant ranges from about 400 pM to about 10 nM, and can optionallycomprise any value within the range, e.g. about 500 pM, about 600 pM,about 700 pM, about 800 pM, about 900 pM, about 1 nM, about 2.5 nM, orabout 5 nM. Even more preferably, the K_(d) is established using adouble-filter nitrocellulose filter binding assay such as that disclosedby Wong and Lohman, 1993, Proc. Natl. Acad. Sci. USA 90, 5428-5432.

Referring now to FIGS. 1A-1B, representative sequences of RNA aptamersof the present invention that bind to and inhibit the activity of FIXaare disclosed. In a preferred embodiment of each of the sequences ofFIGS. 1A-1B, all cytidines are 2′-deoxy-2′ fluoro cytidine and alluridines are 2′-deoxy-2′-fluorouridine. Binding data for the aptamers ofFIGS. 1A and 1B are presented in Tables 1 and 2.

TABLE 1 Affinities (expressed as apparent dissociation constants) ofFIX/FIXa Aptamers for FIXa as determined by Double-Filter NitrocelluloseFilter Binding Assays Sequence K_(d) (pM) SEQ ID NO: 9.3 500-700 3 9.19 500-1000 16 9.20 400-600 17 9.25 400-600 19 9.26 400-600 20 9.271500-1800 21 9.28 500-700 22

TABLE 2 Primary sequence of the minimal or truncated FIXa aptamers9.3^(t) and 9.20^(t)* 9.3^(t): (SEQ ID NO: 70) 5′ AUG GGG ACU AUA CCGCGU AAU GCU GCC UCC CCA U 3′ 9.20^(t): (SEQ ID NO: 71) 5′ GGG GAC UAUACC GGC AAU CGU GCA UCC CC 3′

The apparent K_(d) of the 9.20 truncate for FIXa is ˜100-200 nM. Theapparent K_(d) for binding of the 9.3 truncate to FIXa and to FIX isdescribed in Table 3 immediately below.

TABLE 3 Aptamers to Coagulation Factor IXa Aptamer Protein 9.3 9.3^(t)9.3^(tM) Factor IXa 0.65 ± 0.2 nM 0.58 ± 0.05 nM >10 μM Factor IX 3.96 ±0.7 nM 4.7 ± 0.8 nM n.d. Factor VIIa n.d. >5 μM n.d. Factor Xa n.d. >5μM n.d. Factor XIa n.d. >5 μM n.d. APC n.d. >5 μM n.d.

Table 3 shows affinity of 9.3 and related aptamers to FIXa and othercoagulation factors; 9.3 is a full-length aptamer, while 9.3t is a 35nucleotide truncated version of 9.3.

Referring now to FIGS. 18 and 20, an alignment of FIX/FIXA aptamers ofthe present invention is presented, along with a proposed secondarystructure of truncated aptamer 9-3^(t) (SEQ ID NO: 70). As shown inFIGS. 18 and 20, the alignment of the FIX/FIXA aptamers suggest a firststem region S1 followed by a first loop region L1, and then a secondstem region S2 and a second loop region L2. A third loop region L3 isalso suggested. The motif AUA is common in the L1 region and thus,preferably, a FIX/FIXA aptamer of the present invention comprises theAUA motif. Thus AUA motif thus comprises a consensus sequence for thisfamily of aptamers. Additionally, in the 5′ direction, it is preferredthat 2 additional nucleotides are present, and the nucleotides N in the5′ direction can be selected from the group including but not limited toC, U, G, A and identical combinations thereof. Thus, the sequence NNAUAis also a consensus sequence for this family of aptamers.

Continuing with FIGS. 18 and 20, it is also preferred that the S1 regionpreferably comprises about 5 nucleotides at the 5′ end that form basepairs with about 5 base pairs at the 3′ end to maintain the stemstructure. By “pair forming” it is simply meant the ordinarycomplimentary base pair formation found in RNA molecules. To elaborate,the secondary structure of an RNA motif can be represented by contact intwo dimensions between specific nucleotides. The most easily recognizedsecondary structure motifs are comprised of the Watson/Crick base pairsA:U and C:G. Non-Watson/Crick base pairs, often of lower stability, havebeen recognized, and include the pairs G:U, A:C, G:A, and U:U. (Basepairs are shown once; in RNA molecules the base pair X:Y by conventionrepresents a sequence in which X is 5′ to Y, whereas the base pair Y:Xis also allowed.) A conventional nomenclature for the secondarystructures thus includes stems, loops, hairpin loops, asymmetric bulgedhairpin loops, symmetric hairpin loops, and pseudoknots.

II.B. Coagulation Factor VII Aptamers

In another embodiment, an RNA aptamer of the present inventionselectively binds activated coagulation factor VII (FVIIa) or aninactive form thereof, i.e. (Factor VII). Preferably, the dissociationconstant ranges from about 100 pM to about 10 nM. More preferably, thedissociation constant ranges from about 400 pM to about 10 nM, and canoptionally comprise any value within the range, e.g. about 500 pM, about600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 nM, about 2.5nM, or about 5 nM. Even more preferably, the K_(d) is established usinga double-filter nitrocellulose filter binding assay such as thatdisclosed by Wong and Lohman, 1993, Proc. Natl. Acad. Sci. USA 90,5428-5432.

Referring now to FIG. 10, representative sequences of RNA aptamers ofthe present invention that bind to and inhibit the activity ofcoagulation factor FVIIa are disclosed. In a preferred embodiment ofeach of the sequences of FIG. 10, all cytidines are 2′-deoxy-2′aminocytidine and all uridines are 2′-deoxy-2′-aminouridine. Bindingdata for the aptamers of FIG. 10 are presented in the LaboratoryExamples.

Table 4 shows additional sequences of RNA aptamers isolated tocoagulation factor VIIa. Shown are the random-region derived sequences(5′ to 3′) of aptamers resulting from the selection against FVIIa. In apreferred embodiment, all pyrimidines are 2′fluoro-modified. The randomregion derived sequences are flanked by fixed sequences from the libraryas shown: 5′ gggagagaggaagagggauggg (SEQ ID NO:102)-randomregion-cauaacccagaggucgau 3′ (SEQ ID NO:103). Binding data for theaptamers of Table 4 are presented in the Laboratory Examples. In Table4, the numerals “10” and “11” refer to the fact the aptamer was isolatedafter ten (10) rounds or eleven (11) rounds of SELEX, respectively.

TABLE 4 FVII/FVIIa Aptamers SEQ ID SELEX ROUND NO: AND SPECIE RandonRegion of Aptamer Sequence 74 11-15AAAGUACCGACUAGGUCCCACUGUUUAAGCAUCCCCGAAC 75 10-15AAGCUCCAUCCAAGCGACGACACGCUCGUCCCGAAAAGAAU 76 10-1, 10-5,AAGCUCCGUCCAAGCGACGACACGUUCGUCCCGAAAAGAAU 11-19 77 11-6, 11-27ACAACGCCACCUUCCGCGCGACGCCGCGCCGACGAUAACU 78 10-7ACAACGCCACCUUCCGCGCGACGCCGCGCCGACGUAUAACU 79 11-1ACGAAAAUAUCUCCGUCAAGGACCUCCUGCCCCAAACACU 80 11-17AGACGACACAUCCAAGCGUGAGAGAUCACCCGACAAGAAU 81 11-20AUUUUUUCACACAUUCUUAAUUUUCACUUACCCGUCCCGAUC 82 10-9CAAAGCACCCGUCCAAGCGACAGACAUGUCCCGCAGCCCU 83 10-13CACCAUUUAUUCUUCAUUUUUCUUCGCCCAGUUCCUCCAA 84 10-14CAUAAGCCGCCUCAGCUGACAAAGCCCUCCGCUUAGGCC 85 11-23CCAAAGUGCUUCCGCGAAGUUCGACCAUUCGCCGCCUGCA 86 10-11CCCCUCCGCCAACUUGGCCGCCUCAGGCACCAUCACCAAC 87 10-2CCCGAUCUCCCCGAGGACCUCCACGGCCCGUCCGCCAGUUU 88 11-3CCGCCUCAGCAAUCUAGCCCUCCGCCCGACCCUUCCGCUG 89 11-12, 11-13CCGCCUCAGCGAGAUCUUCGCCCUCCGCCCAAGCCUCAAC 90 11-25CCGCCUCAGGACGACACCGGUCCCCUCCGCCCGUCCGCGC 91 10-3CCGCCUCAGGCAUCAGCCCCUCCGCCCGCCCACUUCAUCA 92 10-12CCGCCUCAGUUACUUGAUAACCCUCCGCCCGCCCGCAGCU 93 11-18CUUUACAUAUUACUUACUACAUUUUCAUAACACCACACGC 94 11-7GACACCAUCCAAGCGACCAACCAAGGUCCCGCACAUAACU 95 10-10GAUGCAACUCGAAAUGGCCGCCUCGCGUCAGCGUUCCGC 96 10-4GCUUAUCUUAUAUCACUUUUUCUUCCCAAUCCUUCAAGU 97 10-6, 98 11-5UAACCAACCAAGCGUCCAAAAACCUGGACCCGCCAAGAAU 99 11-14UAACCAACCAAGCGUCCAAAAAUCUGGACCCGCCAAGAAU 100 11-10UCUGACGUUCCACCGUCCUCGAAGGCGACCAGAGCGUUAC 101 11-8, 11-16UGCCGCCUCAGCCACACGGCCCUCCGCGCCCGCCACAAGC

II.C. Coagulation Factor X Aptamers

In another embodiment, an RNA aptamer of the present inventionselectively binds activated coagulation factor Xa (FXa) or an inactiveform thereof, i.e. (Factor X). Preferably, the dissociation constantranges from about 100 pM to about 10 nM. More preferably, thedissociation constant ranges from about 400 pM to about 10 nM, and canoptionally comprise any value within the range, e.g. about 500 pM, about600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 nM, about 2.5nM, or about 5 nM. Even more preferably, the K_(d) is established usinga double-filter nitrocellulose filter binding assay such as thatdisclosed by Wong and Lohman, 1993, Proc. Natl. Acad. Sci. USA 90,5428-5432.

Referring now to FIGS. 6A-6B, representative sequences of RNA aptamersof the present invention that bind to and inhibit the activity of FXaare disclosed. In a preferred embodiment of each of the sequences ofFIGS. 6A-6B, all cytidines are 2′-deoxy-2′ fluorocytidine and alluridines are 2′-deoxy-2′-fluorouridine. Binding data for the aptamers ofFIGS. 6A and 6B are presented in the Laboratory Examples.

Table 5 shows additional sequences of RNA aptamers isolated tocoagulation factor X/Xa. Binding data for the aptamers of Table 5 arepresented in the Laboratory Examples, including Example 12. In Table 5,the numerals “10” and “11” refer to the fact the aptamer was isolatedafter ten (10) rounds or eleven (11) rounds of SELEX, respectively. TheFXa sequences listed in table 5 are flanked by fixed sequences that arethe same as the fixed sequences described in the preceding section onaptamers to factor VIIa. That is, the 5′ and 3′ flanking sequences are,respectively, SEQ ID NOs:102 and 103.

TABLE 5 SEQ ID SELEX Round And Specie No. Kd. NO: Clone Sequence (nM)104 F11-8 AgAuuAGCCCCAGCGAGAuAAuACuuGGCCCCGCuACuAC 2.2 105 D7-9uAAAuAGCCCCAGCGAGAuucuACuuGGCCCCGCuACuAC 2.633 106 D6-6AAAAuAcgCCannCGAGAuuAuACuuGGCCCCGCuAauAC 3.1 107 F11-7AAAAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCuAuuAC 3.6 108 D7-6AAAAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCuAgcAC 3.8 109 D6-2AAAAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCuACaAC 4.6 110 D7-1AgAAugGCCCCAGCGAGAuuAuACuuGGCCCCGCcAauAC 4.7 111 D6-1AAAAuAGCCCCAGCGAGAugAuACuuGGCCCCGCuAauAC 4.8 112 D7-7AgAAuAcgCCuAGCGAGAagAuACuuGGCCCCCGugCaAC 5.8 113 D6-3AAAAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCuguuAC 5.9 114 D6-9AAAuuuGCCCCAGCGAGAuAAuACuuGGCCCCGCaACuAC 6.5 115 D4-7AuAAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCuACuAa similar to 10-14 116 D7-2AgAAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCuAauAC 8.1 117 D6-4AAAuuuGCCCuAGCGAGAuuAuACuuGGCCCCGCgAaaaAC 8.5 118 E10-12AAAAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCgAacAC 11.7 119 D6-7ugcAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCuACaAC 13.2 120 D7-8ngAuuAGCCCnAGCGAGAuAnuACuuGGCCCCGCuACnuC 14.49 121 D4-8AAAAuAaCCaCAGCGAGAucAuACuuGGCCCCGuuACuAC 2 fold higher 122 D4-10AAAAuAGCCCuAGCGAGAuAAuACuuGGCCCCGCcACaua 2 fold higher 123 D6-10cAgAuAGCCaCAGCGAGAucAuACuuGGCCCCGCuACuAC 18.7 124 D4-5AgAAuAGCCCCAGCGAGAuAAucCuuGGCCCCGCuACugC 21.66 125 D4-6AAncuAGCCCnAGCGAGAuAuuACuuGGCCCCGCnACuAC 3 fold higher 126 D7-5AAAcuAGCCuCAGCGAGAuAAuACuuGGCCCCGCuACuAC 26.1 127 E10-11CCAGAAGCGCuCACuACAACGuuGAACCCCCCGuCCACAC 27.11 128 E10-8CCAAAAGCGGACuGAAGACGuGuuuCCCCCAuCuCCGuGA 28.38 129 E9-8CCAGAAGGAACuAAACACCuGAACCCCCCAuCGCGAGAGA 29.81 130 F11-6CCAGCAACGuCACACGAACGGAAuACCCCCCAuuGAAAAC 33.6 131 E10-4uCuuAGAuAuAGAACuCCGAGAGGACuGACCGuACAGAAC 37.44 132 D7-4AgAAuAGCCCCAGCGAGAucguACuuGGCCCCGCuAguAC 39.3 133 E9-3CCAAAAGCGCAuACACCuGCGuGuuuCCCCCGCCAACAGu 46.55 134 E10-1CCAuuGCuNCCCuGAACANGGGCNCCACNCCGCCuNCACAGu 51.7 135 E9-9CCAGAACACCAGuGAACCCCCCAGCCCCuuCuCACCAGAu 52.81 136 E9-5CCAGAAGCGACACuAACGCuGAACCCCCCAGuCCCuuCACGuG 53.5 137 E9-2AuACCGAGCACGCAAAACACACAAuGCCCAAGCAGGACu 58.6 138 E9-4AGCCCGAGAAAAuAACGCGuuCCACCAuACuACuAAGC 65 139 D4-3uAAAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCaACuAC 65.5 140 E10-7AGuCCGACuGGAGAACANGuACuCuAuAAGCACuuNCAuNCAN 69.45 141 F11-10CuCGGCAGAAGACACGCAuuCACCuGGuGCCACCuCGuAA 84.43 142 E10-6GCCGuCGCCAGGAAuCAAACuGCuACuCCAuCCCGGGCA 85.7 143 E9-6CCAGAAGCuAAACACuCAuAACCACGCuGAACCCCCCAAC 95.1 144 E9-10CCAGAACCAACuGCGGuGAACCCCCCAuACCGCGACACAu 130.4 145 D4-2AAcuuAGCCuCAGCGAGAuAAcgCuuGGCCCCGCuAagAC 540 146 D4-9uAAguuGCCCCAGCGAGAuAguACuuGGCCCCGCuACuAa >240 147 E9-1AAAAuAGCCCCAGCGAGAuAAuACuuGGCCCCGCuACuAA

II.D. Thrombin Aptamers

In another embodiment, an RNA aptamer of the present inventionselectively binds coagulation factor thrombin (activated) (Factor IIa(FIIa)), or an inactive form thereof (i.e. prothrombin, factor VII).Preferably, the dissociation constant ranges from about 100 pM to about10 nM. More preferably, the dissociation constant ranges from about 400pM to about 10 nM, and can optionally comprise any value within therange, e.g. about 500 pM, about 600 pM, about 700 pM, about 800 pM,about 900 pM, about 1 nM, about 2.5 nM, or about 5 nM. Even morepreferably, the K_(d) is established using a double-filternitrocellulose filter binding assay such as that disclosed by Wong andLohman, 1993, Proc. Natl. Acad. Sci. USA 90, 5428-5432.

Referring now to FIG. 11, representative full-length thrombin aptamersequences are disclosed, along with dissociation constants (K_(d)) forhuman and porcine thrombin. Significantly, 13 rounds of the toggle SELEXmethod disclosed herein in accordance with the present inventionproduced these aptamers. A preferred consensus sequence of theseaptamers comprises AACAA. A preferred embodiment of a thrombin aptamersequences comprises the sequence referred to as toggle 25 (TOG25; SEQ IDNO: 55). A truncated form of this aptamer was also prepared, and thetruncate is referred to herein as TOG 25 short (SEQ ID NO: 57).

As shown in FIG. 15, a thrombin RNA aptamer of the present inventionpreferably comprises about 3 pair forming nucleotides followed by theAACAA consensus sequence and two additional pair forming nucleotides. Asshown in FIG. 15, the pair forming nucleotides form pairs withcomplementary nucleotides proximate to the 3′ end of the TOG 25 shortaptamer.

II.E. E2F Aptamers

In another embodiment, an RNA aptamer of the present inventionselectively binds E2-F, also referred to as “an E2F family member”.Thus, the terms “E2-F” and “E2-F family member” encompass any E2F familymember, whether now known or hereafter identified. Representative E2Ffamily members include but are not limited to E2F-1, E2F-2, E2F-3,E2F-4, E2F-5 and E2F-6. Preferably, the dissociation constant rangesfrom about 100 pM to about 50 nM. More preferably, the dissociationconstant ranges from about 400 pM to about 10 nM, and can optionallycomprise any value within the range, e.g. about 500 pM, about 600 pM,about 700 pM, about 800 pM, about 900 pM, about 1 nM, about 2.5 nM,about 5 nM, or about 10 nM. Even more preferably, the K_(d) isestablished using a double-filter nitrocellulose filter binding assaysuch as that disclosed by Wong and Lohman, 1993, Proc. Natl. Acad. Sci.USA 90, 5428-5432.

Referring now to FIG. 17, representative sequences of RNA aptamers (SEQID NO: 62-69) that bind to and inhibit the activity of E2F familymembers including E2F-3 are disclosed. Each of the aptamers (SEQ ID NO:64-69) are preferably bounded at their 5′ end by the 5 primer sequence5′P of SEQ ID NO: 62 and are preferably bounded at their 3′ end by the 3primer sequence 3′P of (SEQ ID NO: 63). These primers were identifiedafter 10 rounds of SELEX wherein the SELEX method was modified inaccordance with the matrix conditions described in Examples 1 and 2. Theapproximate K_(d)'s for these aptamers are set forth in Table 6.

TABLE 6A Approximate K_(d)'s for E2F Aptamers Aptamer No. SEQ ID NO.K_(d) 10-1 and 10-8 64 1.5 nM 10-2 65 3 nM 10-3, 10-7, 10-11 and 66 1 nM10-12 10-4 67 5 nM 10-5 68 2.5 nM 10-6 69 2 nM

Thus, RNA aptamer ligands that selectively bind to an E2F family member,e.g. E2F-3, are disclosed herein. Among other utilities as disclosedherein, these aptamers have utility in the control of cellproliferation, which is of central importance to the proper developmentof a multi-cellular organism, the homeostatic maintenance of tissues,and the ability of certain cell types to respond appropriately toenvironmental cues. E2F is a family of transcription factors andmodulation of E2F activity is envisioned to be particularly effective inrepeating a wide variety of proliferative events in view of the controlof E2F activity occurring as the end result of G1 cyclin dependentkinase regulatory cascades that involve the Rb family of proteins. See,e.g., Sherr, C. J., Cell, 73:1059-1065 (1993); Hunter, T., Cell75:839-841 (1993); Nevins J. R., Science, 258:424-429 (1992); Helin, K.and Harlow, E., Trends Cell Biol. 3:43-46 (1993); La Thangue, N. B.,Trends Biochem. Sci. 19:180-114 (1994); Sherr, C. J. and Roberts, J. M.,Genes Dev. 9:1149-1143 (1995); Weinberg, R. A. Cell 81:323-330 (1995)Harbour, J. W. and Dean, D. C., Genes and Development 14:2393-2409(2000); and Black, A. R. and Azizkhan-Clifford, J., Gene 237:281-302(1999).

Table 6B shows additional sequences of RNA aptamers isolated to an E2Ffamily member. Shown are the random-region derived sequences (5′ to 3′)of aptamers resulting from the selection against E2F. In a preferredembodiment, all pyrimidines are 2′fluoro-modified. The random regionderived sequences are flanked by fixed sequences from the library asshown: 5′ GGG GGA AUU CUA AUA CGA CUC ACU AUA GGG AGA GAG GAA GAG GGAUGG G (SEQ ID NO:188)-random region-C AUA ACC CAG AGG UCG AUA GUA CUGGAU CCC CCC 3′ (SEQ ID NO:63). Binding data in the form of K_(d) valuesare also shown.

TABLE 6B E2F Aptamers SEQ ID NO: Random Region of Aptamer Sequence K_(d)189 GCUGCCGCGCCUGGACCCCACCCACAUAUGGGCCAC 1.5 nM ACAC 190 AAUGACAAUUGACUCGGAAACCCUCAUGUUCCAAC ACCGG 191CCUACUCUCCACACCUGGUUUUAUGCUCUACACACC UCAC 192CUGCCCCGACCACAAAGGACGGAACCCUACCCACAG UGGG 193CAUAAAAGCAAUUUGCCACCGGCGUACGGCACCCCA AUAU 194CACCUAUGCCAUCAGGCCUCAAUCUCCGGCAGCGAC UCUA 195AUCAACCACAGGAAGAGUGCAGCCAUAGCACACAGA CCA 196GCGACAUACCCCACCCACACUGGCACAACGCGCAAU GCCG 197CUUCAAAGGUCCUGUAUCCAGCCACCCCACUGACAG GA 198CUACCCAGCAAGGUCAACCCUACCCACACUGG 1.75 nM 199AUCUUAAAGAUCACCGGCGUUCGGCAACACCCGACC CAAA 200GCACUAAACUUCGAUUACCCCCCACCCACACUGGCU 4 nM GCAC 201CAGAUUACCCUACCCACACUGCGUGCGGACAACCAU 0.4 nM UGGC 202GCACAAAUGAGAACACGAGUUCACCCCGCCCACACU GGA 203GCGCAGAUCAACCCUACCCAUACUGGGCUCCUUGUG AAGG 204CAAGCGCUGAAACCAAUGCACCCCACCCCACACUGG 1.25 nM UGUAC 205AUGUGAAACACAGAAGCCCUGUACAGACCGCCGACU GUCA* 206CAAACUCACAGACACCAACUGCAGGAGCACCCACCA CGAC 207CGAACGAACUGUGGACCCUACCCACACUGGGCCAAG 0.4 nM CGAU 208CGCCCUGGAACGAGAUUCCUGUAAACCCCCAUCUAG UAGA 209CAAGGUGACCGCGAACCCUACCCGCCGCACGGUAAC AGCG 210CAUCCAGACUACUGGCCCAACCCGCCGCUCCAACCC CGUG 211CUCUCUCCGUAACCAACAAGUCCCAAUGAACAACCA CCAU 212CACUGAACGAAUGGCAACCGCCAAACCCUACCCACA 0.2 nM CUGG* 213CAAGCGUAUACCCUACCCACACUGAGCUACAUUGCG 8 nM CUGA 214GCCGAGAGUGAGUGACCACAACCCCGCCCACACUGG 1.5 nM AAUA 215UUUCCUAUGGCGAUAACUUCAGCCACGCCGGCGCCC 30 nM CGUG 216CGUCACUCCGUCCCAGCCGACGAAGUCCGUAAUUCC 45 nM UCCA 217CCACCCGAAGCAAAUCAAGCCCGACGGCGCUCGGAC 15 nM CAAC 218CGAACUGAAGCUAGCGUAACCCUACCCACACUGCAC 0.5-4 nM GUG 219ACCUCGACCCUUCACCUGACUCUCCCAGAAGUUCUG 2 nM UUUC 220CAAUCCAUACGCACCCGGUCCACACUGGGUUGGAGC 1-40 nM NNN 221AAUGGAAUCACUGAAGGCCCUCCGUAGCACCUAACA 1.5 nM CAGU* 222GCAUCCUGCCAGCGGCGACGGACCUUCGCCCACAGG 2 nM CCUC 223UUAUAUAGCACACUGAAGCCCUCAGCAAAACCUCCA 1.5 nM CAGG* 224UAUGAAAUCACAGAAGCCCGCGUUCGACACCUCC A 3 nM CUGUU* 225CAAACUCACAGACUCCAACUGCAGGAGCACCCACCC 0.25-2.5 nM ACACUGGGACAG 226AUCCCCGCCGUAAGCCGUCCUGAUGGACACCACACG 2 nM CCGC*

II.F. Angiopoietin-1 (Ang1) and Angiopoietin-2 (Ang2) Aptamers

Tie2 is an endothelial receptor tyrosine kinase (RTK) that is requiredfor both embryonic vascular development and pathological angiogenesis.Tie2 is unique among RTKs in that it has two ligands with apparentlyopposing actions. Angiopoietin-1 (Ang1) is an activating ligand whileAngiopoietin-2 (Ang2) is thought to be a naturally occurring antagonistfor Tie2. Mice lacking Tie2 or Ang1 die midway through gestation due toabnormalities of vascular morphogenesis characterized by deficientrecruitment of supporting smooth muscle cells and pericytes. Moreover,Ang1 promotes endothelial cell survival and blocks the increases invascular permeability induced by vascular endothelial growth factor(VEGF), supporting a role for Ang1 in the stabilization and maintenanceof the adult vasculature. In contrast, Ang2 is required forVEGF-mediated angiogenesis, and in the absence of endothelial mitogensAng2 may induce vascular regression.

The exact mechanism of action of the Angiopoietins remains to beelucidated. For example, high-dose Ang2 can induce downstream activationof Akt and endothelial cell survival, suggesting that it does not simplyexert a dominant negative effect on Tie2. The need for improvedunderstanding of these ligands' function is particularly important inthe study of tumor angiogenesis, as several studies have now shown thatinhibition of Tie2 with a soluble receptor blocks tumor growth,angiogenesis, and metastasis. However, it is unclear whether theseeffects are due to inhibition of the effects of Ang1 or Ang2, since asoluble receptor would bind both ligands. Specific inhibitors of theseligands have the potential to more precisely modulate Tie2 signaling andserve as valuable therapeutic agents. The present invention provides RNAaptamer ligands for Ang1 and Ang2.

A random library of 2′-fluoro modified RNA was created with thefollowing sequence: GGGAGAGAGGAAGAGGGAUGGG (SEQ IDNO:149)-N₄₀-CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO:150), whereN₄₀ is a random region.

In vitro selection was performed using recombinant human angiopoietin-1*(ANG1*; Regeneron Pharmaceuticals, Tarrytown, N.Y.). After 9 rounds ofSELEX, the pool was cloned and sequenced, and a single random regionsequence (ANG9-4-SEQ ID NO:151) was identified:

ACUCGAACAUUUCCACUAACCAACCAUACUAAAGCACCGC.

In FIG. 24, direct binding (corrected fraction bound) of ANG9-4 RNA isplotted as a function of protein concentration. ANG9-4 binds Ang1 with aK_(d) of ˜10 nM and binds the related antagonist Ang2 with a K_(d) of >1μM (>100-fold specificity). Additional data pertaining to Ang1 aptamersis presented in the Laboratory Examples.

Table 7 sets forth additional representative Ang1 aptamers. Analysis ofearlier SELEX rounds revealed several unique clones with random regionsequences bearing similarities to ANG9-4. Aptamer sequences are asfollows: GGGAGAGAGGAAGAGGGAUGGG (SEQ ID NO:149)-N₄₀CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO:150).

TABLE 7 Ang1 Aptamers SEQ Clone - SELEX ID round number NO: and specieRandom region (N₄₀) 152 9-4 ACUCGAACAUUUCCACUAACCAACCAUACUAAAG CACCGC153 7-2 GACCACCAACACACCACAUACUGCUUUGUACCAA CAUUC 154 6-1CCCAGCGAACACACAACAGAACACGAACGGAUCC GAGCAA 155 6-2GUCACAAACUACCUUCAUCCUUCGCUUGAUACAA CAUUC 156 6-7ACACCAAGGACCCAACGACCCUCGCUUGACACAG UCAUUC 157 6-8AUGAACAACACCCAAACUUGCUUCAACCGCAUCC ACA 158 6-11GACCUCACGCACUGCUAAGCGGCUCUGAUGGAGC UCUAUG 159 6-13CCACCUCCGAAAAAUCACAAUCUGCCCUUGACAC CAGCUAG 160 6-17CCUCAUUGGCCCUGCCACGCUCGGACAACCGUUC CGCUCA 161 6-21UCCAGUGCAGUUCCAUAACCGCUACUCAGCGCGU GAUUAG 162 6-22UUUCGAGCAACCUCCCAACAAUCUAACCGUAACC CUCCAG 163 6-27CAACAUCAGCACGCCUGAACCUUCGCUUGCAACA GCAUUC 164 6-29CCACCUCCGAAAAAUCACAAUCUGCCCUUGACAC CAGCUAG 165 6-31UUACACCAUCGACCAAACUAUGCGCCGUACCACU AUACGA

For Angiopoietin-2 (Ang2) Aptamers, a random library of 2′-fluoromodified RNA was created with the sequence:

GGGAGGACGAUGCGG (SEQ ID NO:166) -N₄₀-CAGACGACUCGCUGAGGAUCCGAGA. (SEQ IDNO:167)

In vitro selection was performed using recombinant human angiopoietin-2(Ang2; R&D Systems, Minneapolis, Minn.). To enhance the specificity ofthe Ang2 selection, molecules that bound the related agonistAngiopoietin-1* (Ang1*; Regeneron Pharmaceuticals) were discarded(negative selection) prior to rounds of positive selection for Ang2.After 11 rounds, the pool was cloned and sequenced, and a family of RNAswith a 12-nucleotide consensus region (italics) was identified and wasrepresented by one dominant random region sequence (ANG11-1):ACUAGCCUCAUCAGCUCAUG UGCCCCUCCGCCUGGAUCAC (SEQ ID NO:168). As shown inFIG. 27, direct binding (corrected fraction bound) of ANG11-1 RNA isplotted as a function of protein concentration. ANG11-1 binds Ang2 witha K_(d) of <10 nM and binds Ang1* with a K_(d) of ˜1 μM (>100-foldspecificity).

Table 8 sets forth additional representative Ang2 aptamers. Analysis ofclones from rounds 9 and 11 revealed several unique random regionsequences containing at least one of two conserved motifs, indicated byitalics or underlining. Aptamer sequences are as follows:GGGAGGACGAUGCGG (SEQ ID NO:166)-N₄₀-CAGACGACUCGCUGAGGAUCCGAGA (SEQ IDNO:167).

TABLE 8 Ang2 Aptamers Clone - SELEX round SEQ number ID and NO: specieRandom region (N₄₀) Frequency K_(d) 168 11-1ACUAGCCUCAUCAGCUCAUGUGCCCCUCCGCCUGGAUCAC 10 of 26   ~5 nM 169 11-2UGACCAAGCCUCACGUUGAACCUGCCAGUAGACCCCGCCCA 1 of 26 170 11-3UUAACCAUCAGCUCAUGGCCCCUGCCCUCUCAAGGACCAC 1 of 26 ~10 nM 171 11-4CACCAGACCGACAUCAGCUUAUGGCCCCUCACCCACACCG 1 of 26 172 11-6GGAGCGCAAUUCGCCUCGCAAGUUGAACUCCGCUGGCGG 1 of 26 173 11-9UAAGCUCUUUGGCUUAGCCCGACACGUUGAACUCCAGAGU 1 of 26 174 11-10CACGGUACCACCAAGUCACACGUUGAACUCCAUGCAGCUG 1 of 26 175 11-15CCACCGAUCGCAUCAGCUCAUGGCCCCUCCCGACCCGCCA 1 of 26 176 11-19CCAGACGUUCUCGCCCCGCCGAUCAUCAGCGCUGGCCCUAU 1 of 26 177 11-26CACUACCACGCCAUAUCAGCUAAUGGCCCCUCCCUACGCA 1 of 26 178 11-30CACUCAGCGCCCUGCGAAACGUUGCCGCCUCCCAACGUCU 1 of 26 179 11-32ACUCACCAGUCACCAUCAGCUCAUGCGCCCCUCCCCCGAC 1 of 26 180 11-34CUCUUUUUGUCCCCGCACGUUGAACUCCUGUCCCUCUACU 1 of 26 181 11-36UGACGGUUCUUCUCUCGCCUCUGGAGCUCUCGUCUCGAU 2 of 26 182  9-1 CACUUUAGCUCACGCCACCGCACGUUGAACGCCCAUCCCG 1 of 8  183  9-2CAAUGCAGCAUCAGCUCAUGGCCCCUCCACAAGCGCGAAU 1 of 8  184  9-3CAUGUCUACAACAAUCUCGCCCGUUGAGUCUCGUCGAAUU 2 of 8  185  9-5 CGAU CU UUUCGUCAACCGCACGUUGAACUCGGCUCGGCAC 1 of 8   ~5 nM 186  9-6CACCCGUCCGUCCAAAUCCGCUUCGUUGGACCCCAUCUU 1 of 8 

Referring now to FIG. 30, application of sequence alignment andRNA-folding algorithms to the predominant sequence family suggests acommon stem-looped structure that incorporated the conserved sequencemotif. Shown is a truncated form (dubbed 11-1.41) of the above-notedaptamer 11.1 (SEQ ID NO:168). The sequence of 11-1.41 isGAGGACGAUGCGGACUAGCCUCAUCAGCUCAUGUGCCCCUC (SEQ ID NO:187). Scramblingthe 6-nucleotide consensus loop sequence (CAGCUC>ACUCCG) disrupts theability of the truncate to bind Ang2 (K_(d)>1000 nM), as does mutationof the sequence comprising the terminal stem ((K_(d)˜1000 nM).

II.G. General Considerations for Aptamers

When a consensus sequence is identified, oligonucleotides that containthat sequence can be made by conventional synthetic or recombinanttechniques. These aptamers can also function as target-specific aptamersof this invention. Such an aptamer can conserve the entire nucleotidesequence of an isolated aptamer, or can contain one or more additions,deletions or substitutions in the nucleotide sequence, as long as aconsensus sequence is conserved. A mixture of such aptamers can alsofunction as target-specific aptamers, wherein the mixture is a set ofaptamers with a portion or portions of their nucleotide sequence beingrandom or varying, and a conserved region that contains the consensussequence. Additionally, secondary aptamers can be synthesized using oneor more of the modified bases, sugars and linkages described hereinusing conventional techniques and those described herein.

In some embodiments of this invention, aptamers can be sequenced ormutagenized to identify consensus regions or domains that areparticipating in aptamer binding to target, and/or aptamer structure.This information is used for generating second and subsequent pools ofaptamers of partially known or predetermined sequence. Sequencing usedalone or in combination with the retention and selection processes ofthis invention, can be used to generate less diverse oligonucleotidepools from which aptamers can be made. Further selection according tothese methods can be carried out to generate aptamers having preferredcharacteristics for diagnostic or therapeutic applications. That is,domains that facilitate, for example, drug delivery could be engineeredinto the aptamers selected according to this invention.

Although this invention is directed to making aptamers using screeningfrom pools of non-predetermined sequences of oligonucleotides, it alsocan be used to make second-generation aptamers from pools of known orpartially known sequences of oligonucleotides. A pool is considereddiverse even if one or both ends of the oligonucleotides comprising itare not identical from one oligonucleotide pool member to another, or ifone or both ends of the oligonucleotides comprising the pool areidentical with non-identical intermediate regions from one pool memberto another. Toward this objective, knowledge of the structure andorganization of the target protein can be useful to distinguish featuresthat are important for biochemical pathway inhibition or biologicalresponse generation in the first generation aptamers. Structuralfeatures can be considered in generating a second (less random) pool ofoligonucleotides for generating second round aptamers.

Those skilled in the art will appreciate that comparisons of thecomplete or partial amino acid sequences of the purified protein targetto identify variable and conserved regions is useful. Comparison ofsequences of aptamers made according to this invention providesinformation about the consensus regions and consensus sequencesresponsible for binding. It is expected that certain nucleotides will berigidly specified and certain positions will exclusively require certainbases. Likewise, studying localized regions of a protein to identifysecondary structure can be useful. Localized regions of a protein canadopt a number of different conformations including beta strands, alphahelices, turns (induced principally by proline or glycine residues) orrandom structure. Different regions of a polypeptide interact with eachother through hydrophobic and electrostatic interactions and also byformation of salt bridges, disulfide bridges, etc. to form the secondaryand tertiary structures. Defined conformations can be formed within theprotein organization, including beta sheets, beta barrels, and clustersof alpha helices.

It sometimes is possible to determine the shape of a protein target orportion thereof by crystallography X ray diffraction or by otherphysical or chemical techniques known to those skilled in the art. Manydifferent computer programs are available for predicting proteinsecondary and tertiary structure, the most common being those describedin Chou, P. Y. and Fasman, G. D. (1978) Biochemistry, 13:222-245, andGamier et al. (1978) J. Mol. Biol., 120:97-120. Generally, these andother available programs are based on the physical and chemicalproperties of individual amino acids (hydrophobicity, size, charge andpresence of side chains) and on the amino acids' collective tendency toform identifiable structures in proteins whose secondary structure hasbeen determined. Many programs attempt to weight structural data withtheir known influences. For example, amino acids such as proline orglycine are often present where polypeptides have share turns. Longstretches of hydrophobic amino acids (as determined by hydropathy plot),usually have a strong affinity for lipids.

Data obtained by the methods described above and other conventionalmethods and tools can be correlated with the presence of particularsequences of nucleotides in the first and second generation aptamers toengineer second and third generation aptamers. Further, according tothis invention, second generation aptamers can be identified simply bysequentially screening from pools of oligonucleotides having morepredetermined sequences than the pools used in earlier rounds ofselection.

These methods can be used to design optimal binding sequences for anydesired protein target (which can be portions of aptamers or entireaptamers) and/or to engineer into aptamers any number of desiredtargeted functions or features. Optimal binding sequences will be thosewhich exhibit high relative affinity for target, i.e., affinity measuredin K_(d) in at least in the nanomolar range, and, for certain drugapplications, the nanomolar or picomolar range. In practicing thisinvention, studying the binding energies of aptamers using standardmethods known generally in the art can be useful generally, consensusregions can be identified by comparing the conservation of nucleotidesfor appreciable enhancement in binding.

Structural knowledge can be used to engineer aptamers made according tothis invention. For example, stem structures in the aptamer pool can bevital components in some embodiments where increased aptamer rigidity isdesired. According to the teachings of the instant invention, a randomlygenerated pool of oligonucleotides having the stem sequences can begenerated. After aptamers are identified which contain the stem (i.e.,use the stem in primers), one can put cross-linkers in the stem tocovalently fix the stem in the aptamer structure. Cross-linkers also canbe used to fix an aptamer to a target.

Once an aptamer has been identified, it can be used, either by linkageto, or use in combination with, other aptamers identified according tothese methods. One or more aptamers can be used in this manner to bindto one or more targets.

II.G.1. Techniques for Identifying Improved Nucleic Acid Ligands

In order to produce nucleic acids desirable for use as a pharmaceutical,it is preferred that the nucleic acid ligand binds to the target in amanner capable of achieving the desired effect on the target; be assmall as possible to obtain the desired effect; be as stable aspossible; and be a specific ligand to the chosen target. In most, if notall, situations it is preferred that the nucleic acid ligand has thehighest possible affinity to the target. Modifications orderivatizations of the ligand that confer resistance to degradation andclearance in situ during therapy, the capability to cross various tissueor cell membrane barriers, or any other accessory properties that do notsignificantly interfere with affinity for the target molecule can alsobe provided as improvements.

One of the uses of nucleic acid ligands derived by SELEX or anotherapproach is to find ligands that alter target molecule function. Thus,it is a good procedure to first assay for inhibition or enhancement offunction of the target protein. One could even perform such functionaltests of the combined ligand pool prior to cloning and sequencing.Assays for the biological function of the chosen target are generallyavailable and known to those skilled in the art, and can be easilyperformed in the presence of the nucleic acid ligand to determine ifinhibition occurs.

Enrichment will supply a number of cloned ligands of probable variableaffinity for the target molecule. Sequence comparisons can yieldconsensus secondary structures and primary sequences that allow groupingof the ligand sequences into motifs. Although a single ligand sequence(with some mutations) can be found frequently in the total population ofcloned sequences, the degree of representation of a single ligandsequence in the cloned population of ligand sequences cannot absolutelycorrelate with affinity for the target molecule. Therefore mereabundance is not the sole criterion for judging “winners” after SELEXand binding assays for various ligand sequences (adequately definingeach motif that is discovered by sequence analysis) are required toweigh the significance of the consensus arrived at by sequencecomparisons. The combination of sequence comparison and affinity assaysshould guide the selection of candidates for more extensive ligandcharacterization.

An important avenue for narrowing down what amount of sequence isrelevant to specific affinity is to establish the boundaries of thatinformation within a ligand sequence. This is conveniently accomplishedby selecting end-labeled fragments from hydrolyzed pools of the ligandof interest so that 5′ and 3′ boundaries of the information can bediscovered. To determine a 3′ boundary, one can perform a large-scale invitro transcription of the PCR'd ligand, gel purifies the RNA using UVshadowing on an intensifying screen, phosphatases the purified RNA,phenol extracts extensively, labels by kinasing with ³²P, and gelpurifies the labeled product (using a film of the gel as a guide). Theresultant product can then be subjected to pilot partial digestions withRNase T1 (varying enzyme concentration and time, at 50° C. in a bufferof 7M urea, 50 mM sodium citrate pH 5.2) and alkaline hydrolysis (at 50mM NaCO₃, adjusted to pH 9.0 by prior mixing of 1M bicarbonate andcarbonate solutions; test over ranges of 20 to 60 minutes at 95° C.).Once optimal conditions for alkaline hydrolysis are established (so thatthere is an even distribution of small to larger fragments) one canscale up to provide enough material for selection by the target (usuallyon nitrocellulose filters).

One then sets up binding assays, varying target protein concentrationfrom the lowest saturating protein concentration to that proteinconcentration at which approximately 10% of RNA is bound as determinedby the binding assays for the ligand. One should vary targetconcentration (if target supplies allow) by increasing volume ratherthan decreasing absolute amount of target; this provides a good signalto noise ratio as the amount of RNA bound to the filter is limited bythe absolute amount of target. The RNA is eluted as, for example, inSELEX and then run on a denaturing gel with T1 partial digests so thatthe positions of hydrolysis bands can be related to the ligand sequence.

The 5′ boundary can be similarly determined. Large-scale in vitrotranscriptions are purified as described above. There are two methodsfor labeling the 3′ end of the RNA. One method is to kinase Cp with ³²P(or purchase ³²P-Cp) and ligate to the purified RNA with RNA ligase. Thelabeled RNA is then purified as above and subjected to very identicalprotocols. An alternative is to subject unlabeled RNAs to partialalkaline hydrolyses and extend an annealed, labeled primer with reversetranscriptase as the assay for band positions. One of the advantagesover pCp labeling is the ease of the procedure, the more completesequencing ladder (by dideoxy chain termination sequencing) with whichone can correlate the boundary, and increased yield of assayableproduct. A disadvantage is that the extension on eluted RNA sometimescontains artifactual stops, so it can be important to control byspotting and eluting starting material on nitrocellulose filters withoutwashes and assaying as the input RNA.

The result is that it is possible to find the boundaries of the sequenceinformation required for high affinity binding to the target.

II.G.2. Assessment of Nucleotide Contributions to Affinity

Once a minimal high affinity ligand sequence is identified, it can beuseful to identify the nucleotides within the boundaries that arecrucial to the interaction with the target molecule. One method is tocreate a new random template in which all of the nucleotides of a highaffinity ligand sequence are partially randomized or blocks ofrandomness are interspersed with blocks of complete randomness for usein a SELEX method for example, preferably a modified or toggle SELEXmethod as disclosed herein. Such “secondary” SELEXes produce a pool ofligand sequences in which crucial nucleotides or structures areabsolutely conserved, less crucial features preferred, and unimportantpositions unbiased. Secondary SELEXes can thus help to further elaboratea consensus that is based on relatively few ligand sequences. Inaddition, even higher-affinity ligands can be provided whose sequenceswere unexplored in the original SELEX.

Another method is to test oligo-transcribed variants (i.e. nucleotidesubstitution) where the consensus sequence can be unclear.

Another useful set of techniques are inclusively described as chemicalmodification experiments. Such experiments can be used to probe thenative structure of RNAs, by comparing modification patterns ofdenatured and non-denatured states. The chemical modification pattern ofan RNA ligand that is subsequently bound by target molecule can bedifferent from the native pattern, indicating potential changes instructure upon binding or protection of groups by the target molecule.In addition, RNA ligands will fail to be bound by the target moleculewhen modified at positions crucial to either the bound structure of theligand or crucial to interaction with the target molecule. Suchexperiments in which these positions are identified are described as“chemical modification interference” experiments.

There are a variety of available reagents to conduct such experimentsthat are known to those skilled in the art (see, Ehresmann et al., Nuc.Acids. Res., 15:9109-9128, (1987)). Chemicals that modify bases can beused to modify ligand RNAs. A pool is bound to the target at varyingconcentrations and the bound RNAs recovered (much as in the boundaryexperiments) and the eluted RNAs analyzed for the modification. An assaycan be by subsequent modification-dependent base removal and anilinescission at the baseless position or by reverse transcription assay ofsensitive (modified) positions. In such assays bands (indicatingmodified bases) in unselected RNAs appear that disappear relative toother bands in target protein-selected RNAs. Similar chemicalmodifications with ethylnitrosourea, or via mixed chemical or enzymaticsynthesis with, for example, 2′-methoxys on ribose or phosphorothioatescan be used to identify essential atomic groups on the backbone. Inexperiments with 2′-methoxy vs. 2′-OH mixtures, the presence of anessential OH group results in enhanced hydrolysis relative to otherpositions in molecules that have been stringently selected by thetarget.

Comparisons of the intensity of bands for bound and unbound ligands canreveal not only modifications that interfere with binding, but alsomodifications that enhance binding. A ligand can be made with preciselythat modification and tested for the enhanced affinity. Thus chemicalmodification experiments can be a method for exploring additional localcontacts with the target molecule, just as “walking” (see below) is foradditional nucleotide level contacts with adjacent domains.

A consensus of primary and secondary structures that enables thechemical or enzymatic synthesis of oligonucleotide ligands whose designis based on that consensus is provided herein via a SELEX method,preferably a modified or toggle SELEX method as disclosed herein.Because the replication machinery of SELEX requires that rather limitedvariation at the subunit level (ribonucleotides, for example), suchligands imperfectly fill the available atomic space of a targetmolecule's binding surface. However, these ligands can be thought of ashigh-affinity scaffolds that can be derivatized to make additionalcontacts with the target molecule. In addition, the consensus containsatomic group descriptors that are pertinent to binding and atomic groupdescriptors that are coincidental to the pertinent atomic groupinteractions. Such derivatization does not exclude incorporation ofcross-linking agents that will give specifically directly covalentlinkages to the target protein. Such derivatization analyses can beperformed at but are not limited to the 2′ position of the ribose, andthus can also include derivatization at any position in the base orbackbone of the nucleotide ligand.

The present invention thus includes nucleic acid ligands wherein certainchemical modifications have been made in order to increase the in vivostability of the ligand or to enhance or mediate the delivery of theligand. Examples of such modifications include chemical substitutions atthe ribose and/or phosphate positions of a given RNA sequence. See,e.g., Cook et al., PCT Application WO 9203568; U.S. Pat. No. 5,118,672to Schinazi et al.; Hobbs et al., Biochem 12:5138 (1973); Guschlbauer etal., Nucleic Acids Res. 4:1933 (1977); Shibahara et al., Nucl. Acids.Res. 15:4403 (1987); Pieken et al., Science 253:314 (1991), each ofwhich is specifically incorporated herein by reference.

A logical extension of this analysis is a situation in which one or afew nucleotides of the polymeric ligand is used as a site for chemicalderivative exploration. The rest of the ligand serves to anchor in placethis monomer (or monomers) on which a variety of derivatives are testedfor non-interference with binding and for enhanced affinity. Suchexplorations can result in small molecules that mimic the structure ofthe initial ligand framework, and have significant and specific affinityfor the target molecule independent of that nucleic acid framework. Suchderivatized subunits, which can have advantages with respect to massproduction, therapeutic routes of administration, delivery, clearance ordegradation than the initial ligand, can become the therapeutic and canretain very little of the original ligand. Thus, the aptamer ligands ofthe present invention can allow directed chemical exploration of adefined site on the target molecule known to be important for the targetfunction.

II.G.3. Walking Experiments

After a minimal consensus ligand sequence has been determined for agiven target, it is possible to add random sequence to the minimalconsensus ligand sequence and evolve additional contacts with thetarget, perhaps to separate but adjacent domains. This procedure hasbeen referred to in the art as “walking”.

A walking experiment can involve two experiments performed sequentially.A new candidate mixture is produced in which each of the members of thecandidate mixture has a fixed nucleic acid region that corresponds to anucleic acid ligand of interest. Each member of the candidate mixturealso contains a randomized region of sequences. According to this methodit is possible to identify what are referred to as “extended” nucleicacid ligands, which contain regions that can bind to more than onebinding domain of a target.

II.G.4. Covariance Analysis

In conjunction with empirical methods for determining the threedimensional structure of nucleic acids, computer modeling methods fordetermining structure of nucleic acid ligands can also be employed.

Secondary structure prediction is a useful guide to correct sequencealignment. It is also a highly useful stepping-stone to correct 3Dstructure prediction, by constraining a number of bases into A-formhelical geometry.

Tables of energy parameters for calculating the stability of secondarystructures exist. Although early secondary structure prediction programsattempted to simply maximize the number of base-pairs formed by asequence, most current programs seek to find structures with minimalfree energy as calculated by these thermodynamic parameters. There aretwo problems in this approach that should be borne in mind. First, thethermodynamic rules are inherently inaccurate, typically to 10% or so,and there are many different possible structures lying within 10% of theglobal energy minimum. Second, the actual secondary structure need notlie at a global energy minimum, depending on the kinetics of folding andsynthesis of the sequence. Nonetheless, for short sequences, thesecaveats are of minor importance because there are so few possiblestructures that can form.

The brute force predictive method is a dot-plot: make an N by N plot ofthe sequence against itself, and mark an X everywhere a base pair ispossible. Diagonal runs of X's mark the location of possible helices.Exhaustive tree-searching methods can then search for all possiblearrangements of compatible (i.e., non-overlapping) helices of length Lor more; energy calculations can be done for these structures to rankthem as more or less likely. The advantages of this method are that allpossible topologies, including pseudoknotted conformations, can beexamined, and that a number of suboptimal structures are automaticallygenerated as well. The disadvantages of the method are that it can runin the worst cases in time proportional to an exponential factor of thesequence size, and cannot (depending on the size of the sequence and theactual tree search method employed) look deep enough to find a globalminimum.

An elegant predictive method, and currently the most used, is the Zukerprogram. Zuker (1989) Science 244:48-52. Originally based on analgorithm developed by Ruth Nussinov, the Zuker program makes a majorsimplifying assumption that no pseudoknotted conformations will beallowed. This permits the use of a dynamic programming approach thatruns in time proportional to only N3 to N4, where N is the length of thesequence. The Zuker program is the only program capable of rigorouslydealing with sequences of than a few hundred nucleotides, so it has cometo be the most commonly used by biologists. However, the inability ofthe Zuker program to predict pseudoknotted conformations is a seriousconsideration. Where pseudoknotted RNA structures are suspected or arerecognized by eye, a brute-force method capable of predictingpseudoknotted conformations should be employed.

A central element of comparative sequence analysis is sequencecovariations. A covariation is when the identity of one position dependson the identity of another position; for instance, a requiredWatson-Crick base pair shows strong covariation in that knowledge of oneof the two positions gives absolute knowledge of the identity at theother position. Covariation analysis has been used previously to predictthe secondary structure of RNAs for which a number of related sequencessharing a common structure exist, such as tRNA, rRNAs, and group Iintrons. It is now apparent that covariation analysis can be used todetect tertiary contacts as well.

Stormo and Gutell (1992) Nucleic Acids Research 29:5785-5795 havedesigned and implemented an algorithm that precisely measures the amountof covariations between two positions in an aligned sequence set. Theprogram is called “MIXY”—Mutual Information at position X and Y.Consider an aligned sequence set. In each column or position, thefrequency of occurrence of A, C, G, U, and gaps is calculated. Call thisfrequency f(bx), the frequency of base b in column x. Now consider twocolumns at once. The frequency that a given base b appears in column xis f(bx) and the frequency that a given base b appears in column y isf(by). If position x and position y do not care about each other'sidentity—that is, the positions are independent; there is nocovariation—the frequency of observing bases bx and by at position x andy in any given sequence should be just f(bxby)=f(bx)f(by). If there aresubstantial deviations of the observed frequencies of pairs from theirexpected frequencies, the positions are said to covary.

The amount of deviation from expectation can be quantified with aninformation measure M(x,y), the mutual information of x and y:

${M\left( {x,y} \right)} = {\sum\limits_{b_{x}b_{y}}\; {{f\left( {b_{x}b_{y}} \right)}\; \ln \frac{f\left( {b_{x}b_{y}} \right)}{{f\left( b_{x} \right)}{f\left( b_{y} \right)}}}}$

M(x,y) can be described as the number of bits of information one learnsabout the identity of position y from knowing just the identity ofposition y from knowing just the identity of position x. If there is nocovariation, M(x,y) is zero; larger values of M(x,y) indicate strongcovariation. Covariation values can be used to develop three-dimensionalstructural predictions.

In some ways, the problem is similar to that of structure determinationby NMR. Unlike crystallography, which in the end yields an actualelectron density map, NMR yields a set of interatomic distances.Depending on the number of interatomic distances one can get, there canbe one, few, or many 3D structures with which they are consistent.Mathematical techniques had to be developed to transform a matrix ofinteratomic distances into a structure in 3D space. The two maintechniques in use are distance geometry and restrained moleculardynamics.

Distance geometry is the more formal and purely mathematical technique.The interatomic distances are considered to be coordinates in anN-dimensional space, where N is the number of atoms. In other words, the“position” of an atom is specified by N distances to all the otheratoms, instead of the three (x,y,z) coordinates typically considered.Interatomic distances between every atom are recorded in an N-by-Ndistance matrix. A complete and precise distance matrix is easilytransformed into a 3 by N Cartesian coordinates, using matrix algebraoperations. The trick of distance geometry as applied to NMR is dealingwith incomplete (only some of the interatomic distances are known) andimprecise data (distances are known to a precision of only a fewangstroms at best). Much of the time of distance geometry-basedstructure calculation is thus spent in pre-processing the distancematrix, calculating bounds for the unknown distance values based on theknown ones, and narrowing the bounds on the known ones. Usually,multiple structures are extracted from the distance matrix that areconsistent with a set of NMR data; if they all overlap nicely, the datawere sufficient to determine a unique structure. Unlike NMR structuredetermination, covariance gives only imprecise distance values, but alsoonly probabilistic rather than absolute knowledge about whether a givendistance constraint should be applied.

Restrained molecular dynamics can also be employed, albeit in a more adhoc manner. Given an empirical force field that attempts to describe theforces that all the atoms feel (van der Weals, covalent bonding lengthsand Angles, electrostatics), one can simulate a number of femtosecondtime steps of a molecule's motion, by assigning every atom at a randomvelocity (from the Boltzmann distribution at a given temperature) andcalculating each atom's motion for a femtosecond using Newtoniandynamical equations; that is “molecular dynamics”. In restrainedmolecular dynamics, one assigns extra ad hoc forces to the atoms whenthey violate specified distance bounds.

With respect to RNA aptamers, the probabilistic nature of data withrestrained molecular dynamics can be addressed. The covariation valuescan be transformed into artificial restraining forces between certainatoms for certain distance bounds; varying the magnitude of the forceaccording to the magnitude of the covariance.

NMR and covariance analysis generates distance restraints between atomsor positions, which are readily transformed into structures throughdistance geometry or restrained molecular dynamics. Another source ofexperimental data which can be utilized to determine the threedimensional structures of nucleic acids is chemical and enzymaticprotection experiments, which generate solvent accessibility restraintsfor individual atoms or positions.

II.H. Utility of the Aptamers

The aptamers and modified aptamers of the invention are useful indiagnostic, research and therapeutic contexts. For diagnosticapplications, aptamers are particularly well suited for binding tobiomolecules that are identical or similar between different species.Classes of molecules such as coagulation factors and transcriptionfactors generally do not serve as good antigens because they are noteasily recognized as foreign by the immune systems of animals that canbe used to generate antibodies. Antibodies are generally used to bindanalytes that are detected or quantitated in various diagnostic assays.Aptamers represent a class of molecules that can be used in place ofantibodies for diagnostic and purification purposes.

The aptamers of the invention are therefore particularly useful asdiagnostic reagents to detect the presence or absence of the targetsubstances to which they specifically bind. Such diagnostic tests areconducted by contacting a sample with the specifically bindingoligonucleotide to obtain a complex that is then detected byconventional techniques. For example, the aptamers can be labeled usingradioactive, fluorescent, or chromogenic labels and the presence oflabel bound to solid support to which the target substance has beenbound through a specific or nonspecific binding means detected.Alternatively, the specifically binding aptamers can be used to effectinitial complexation to the support. Techniques for conducting assaysusing such oligomers as specific binding partners are generally known totrack those for standard specific binding partner based assays.

This invention also permits the recovery and deduction of oligomericsequences that bind specifically to target proteins and specificportions thereof. Therefore, these oligonucleotides can be used as aseparation tool for retrieving the substances to which they specificallybind. By coupling the oligonucleotides containing the specificallybinding sequences to a solid support, for example, proteins or othercellular components to which they bind can be recovered in usefulquantities. In addition, these oligonucleotides can be used in diagnosisby employing them in specific binding assays for the target substances.When suitably labeled using detectable moieties such as radioisotopes,the specifically binding oligonucleotides can also be used for in vivoimaging or histological analysis.

It can be commented that the mechanism by which the specifically bindingoligomers of the invention interfere with or inhibit the activity of atarget substance is not always established, and is not a part of theinvention. The oligomers of the invention are characterized by theirability to target specific substances regardless of the mechanisms oftargeting or the mechanism of the effect thereof.

For use in research, the specifically binding oligonucleotides of theinvention are especially helpful in effecting the isolation andpurification of substances to which they bind. For this application,typically, the oligonucleotide containing the specific binding sequencesis conjugated to a solid support and used as an affinity ligand inchromatographic separation of the target substance. The affinity ligandcan also be used to recover previously unknown substances from sourcesthat do not contain the target substance by virtue of binding similaritybetween the intended target and the unknown substances. Furthermore, asdata accumulate with respect to the nature of thenonoligonucleotide/oligonucleotide-specific binding, insight can begained as to the mechanisms for control of gene expression.

The aptamers described herein can be used as a separation reagent forretrieving the targets to which they specifically bind. By coupling theoligonucleotides containing the specifically binding sequences to asolid support, for example, the target substances can be recovered inuseful quantities. In addition, these oligonucleotides can be used indiagnosis by employing them in specific binding assays for the targetsubstances. When suitably labeled using detectable moieties includingradioisotopes such as ¹⁹F, ^(99M)Tc, ¹³¹1, ⁹⁰Y, ¹¹¹1n, ¹²³1, ¹⁵N, ³² or³³P, the specifically binding oligonucleotides can also be used for invivo or in vitro diagnosis, imaging or histological analysis bytechniques known in the art.

For application in such various uses, the aptamers of the invention canbe coupled to auxiliary substances that enhance or complement thefunction of the aptamer. Such auxiliary substances include, for example,labels such as radioisotopes, fluorescent labels, enzyme labels and thelike; specific binding reagents such as antibodies, additional aptamersequence, cell surface receptor ligands, receptors per se and the like;toxins such as diphtheria toxin, tetanus toxin or ricin; drugs such asantiinflammatory, antibiotic, or metabolic regulator pharmaceuticals,solid supports such as chromatographic or electrophoretic supports, andthe like. Suitable techniques for coupling of aptamers to desiredauxiliary substances are generally known for a variety of such auxiliarysubstances, and the specific nature of the coupling procedure willdepend on the nature of the auxiliary substance chosen. Coupling can bedirect covalent coupling or can involve the use of synthetic linkerssuch as those marketed by Pierce Chemical Co., Rockford, Ill.

Thus, the aptamers or modified aptamers of the invention can be usedalone in therapeutic applications or can be used as targeting agents todeliver pharmaceuticals or toxins to desired targets. The aptamers canbe used in diagnostic procedures and advantageously in this applicationinclude label. They can be used as reagents to separate target moleculesfrom contaminants in samples containing the target molecules in whichapplication they are advantageously coupled to solid support.

III. SYNTHESIS OF RNA APTAMERS

In a representative embodiment of the present invention, an RNA aptameris synthesized on a solid support column, using conventional techniquessuch as those described by Beaucage et al. (1981) Tetrahedr. Letters22:1859-1862 and Sinha et al., (1984) Nucleosides and Nucleotides3:157-171, both of which are incorporated by reference. The finalDMT-group is removed from the resulting RNA aptamer. Alternately, iflarge-scale synthesis is used, the RNA aptamer can be made by scale-upof the solid support method or the RNA aptamer can be made by usingsolution phase techniques, particularly if the desired end-product is arelatively short oligonucleotide. A starting material for the synthesisprocess can be a 5′-non-tritylated RNA oligoribo-nucleotide or analog ofthe desired primary structure, which preferably can have protectedbases, and which is preferably bound to a solid-support. Anyconventionally used protecting groups can be used. Typically N₆-benzoylis used for adenine, N₄-benzoyl for cytosine, N₂-isobutyryl for guanineand _(N2)-benzoyl for 2-amino purine. Other useful protecting groupsinclude phenoxyacetyl (PAC) and t-butoxyacetyl (TAC). Conveniently, themore base labile protection groups should be used for the synthesis ofthe RNA or analog fragment; those of ordinary skill in the art knowthese groups. Such groups can help to prevent hydrolysis of thegenerated tri- or diphosphates, which are generally quite stable underbasic conditions, but could be subject to some hydrolysis. Otherenvisioned modifications are disclosed in U.S. Pat. No. 6,011,020,incorporated herein by reference, and include but are not limited to theincorporation of bioavailability enhancing molecules such as PEG orcholesterol via a covalent linkage.

A capped RNA or analog of this invention can be of any length, the onlylimit being that of the synthesis technique employed to prepare the RNAor analog. Currently, a preferred length is ranges from approximately 15to 100 bases, but with improvements in synthetic technology the lengthof the oligonucleotide is expected to increase. For purposes of thisinvention, it is preferred that the capped RNA or analog be less thanapproximately 100 bases in length, and preferably less than about 40bases in length.

In addition, nucleoside analogs such as 2′-deoxy, 2′-halo, 2′-amino (notsubstituted or mono- or disubstituted), 2′-mono, di- or trihalomethyl,2′-O-alkyl, 2′-O-halo-substituted alkyl, 2′-alkyl, azido,phosphorothioate, sulfhydryl, methylphosphonate, fluorescein, rhodamine,pyrene, biotin, xanthine, hypoxanthine, 2,6-diamino purine,2-hydroxy-6-mercaptopurine and pyrimidine bases substituted at the6-position with sulfur or 5 position with halo or C₁₋₅ alkyl groups,abasic linkers, 3′-deoxy-adenosine as well as other available “chainterminator” or “non-extendible” analogs (at the 3′-end of the RNA), andthe like can be incorporated during the RNA synthesis. Further, variouslabels such as ³²P or ³³P and the like can likewise be incorporatedduring the synthesis, resulting in novel RNA analogs produced by thisprocess. Other envisioned modifications are disclosed in U.S. Pat. No.6,011,020, incorporated herein by reference, and include but are notlimited to the incorporation of 3′ caps, such an inverted DT cap, or aninverted abasic cap, or combination thereof.

IV. THERAPEUTIC METHODS

A method of modulating the biological activity of a coagulation pathwayfactor is provided in accordance with the present invention. In apreferred embodiment, the method comprises: (a) administering to a warmblooded vertebrate in need thereof an effective amount of an RNA aptamerthat selectively binds a coagulation pathway factor, the RNA aptamerhaving a dissociation constant for the coagulation pathway factor ofabout 20 nM or less; and (b) modulating the biological activity of thecoagulation pathway factor in the warm-blooded vertebrate through theadministering of the RNA aptamer in step (a).

A method of treating cardiovascular disease in a warm-blooded vertebrateis also provided in accordance with the present invention. The methodcomprises administering an effective amount of an RNA aptamer thatselectively binds a coagulation pathway factor, the RNA aptamer having adissociation constant for the coagulation pathway factor of about 20 nMor less, to a vertebrate subject suffering from cardiovascular disease,whereby cardiovascular disease in the vertebrate subject is treated.

A method of modulating E2F activity in a warm-blooded vertebrate inwhich such modulation is desired is also provided. The method comprises:(a) administering to the warm-blooded vertebrate an effective amount ofan RNA aptamer that selectively binds an E2F family member, the RNAaptamer having a dissociation constant for the E2F family member ofabout 20 nM or less; and (b) modulating E2F in the warm-bloodedvertebrate through the administering of the RNA aptamer of step (a).

The patient treated in the present invention in its many embodiments isdesirably a human patient, although it is to be understood that theprinciples of the invention indicate that the invention is effectivewith respect to all vertebrate species, including warm-blood vertebrates(e.g. birds and mammals), which are intended to be included in the term“patient”. In this context, a mammal is understood to include anymammalian species in which treatment of cardiovascular disease isdesirable, particularly agricultural and domestic mammalian species.

Contemplated is the treatment of mammals such as humans, as well asthose mammals of importance due to being endangered (such as Siberiantigers), of economical importance (animals raised on farms forconsumption by humans and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans such ascats and dogs), swine (pigs, hogs, and wild board), ruminants (such ascattle, oxen, sheep, giraffes, deer goats, bison, and camels), andhorses. Also contemplated is the treatment of birds, including thetreatment of those kinds of birds that are endangered, kept in zoos, aswell as fowl, and more particularly domesticated fowl, i.e., poultry,such as turkeys, chickens, ducks, geese, guinea fowl, and the like, asthey are also of economical importance to humans.

The present method for treating cardiovascular disease in a tissuecontemplates contacting a tissue in which cardiovascular disease isoccurring, or is at risk for occurring, with a composition comprising atherapeutically effective amount of an RNA aptamer capable of binding acoagulation factor. Thus, the method comprises administering to apatient a therapeutically effective amount of a physiologicallytolerable composition containing the RNA aptamer.

The dosage ranges for the administration of the RNA aptamer depend uponthe form of the modulator, and its potency, as described further herein,and are amounts large enough to produce the desired effect in whichcoagulation is modulated, which can correspondingly amelioratecardiovascular disease and the symptoms of cardiovascular disease. Thedosage should not be so large as to cause adverse side effects, such ashyperviscosity syndromes, pulmonary edema, congestive heart failure, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The individual physician in the event of anycomplication can also adjust the dosage.

A therapeutically effective amount is an amount of an RNA aptamersufficient to produce a measurable modulation of coagulation in thetissue being treated, i.e., a coagulation-modulating amount, an E2Factivity-modulating amount coagulation, and/or angiogenesis factoractivity (e.g. Ang1 or Ang2 activity)-modulating amount. Modulation ofcoagulation, E2F activity, and/or angiogenesis factor activity (e.g.Ang1 or Ang2 activity) can be measured in situ by immunohistochemistryby methods disclosed in the Laboratory Examples, or by other methodsknown to one skilled in the art. By the term “modulate” and grammaticalvariations thereof, it is intended an increase, decrease, or otheralteration of any or all biological activities or properties a target.

Insofar as a coagulation modulator, E2F modulator, or angiogenesisfactor activity (e.g. Ang1 or Ang2 activity) modulator can take the formof RNA aptamers it is to be appreciated that the potency, and thereforean expression of a “therapeutically effective” amount can vary. However,as shown by the methods presented in the Laboratory Examples, oneskilled in the art can readily assess the potency of a candidate RNAaptamer of this invention.

A preferred RNA aptamer has the ability to substantially bind to atarget in solution at modulator concentrations of less than one (1)micromolar (μM), preferably less than 0.1 μM, and more preferably lessthan 0.01 μM. By “substantially” is meant that at least a 50 percentreduction in target biological activity is observed by modulation in thepresence of the a target, and at 50% reduction is referred to herein asan IC₅₀ value.

The RNA aptamers of the present invention can be administeredparenterally by injection or by gradual infusion over time. Although thetissue to be treated can typically be accessed in the body by systemicadministration and therefore most often treated by intravenousadministration of therapeutic compositions, other tissues and deliverytechniques are provided where there is a likelihood that the tissuetargeted contains the target molecule. Thus, an RNA aptamer of thepresent invention can be administered orally, topically to a vasculartissue, intravenously, intraperitoneally, intramuscularly,subcutaneously, intra-cavity, transdermally, and can be delivered byperistaltic techniques. Representative, non-limiting approaches fortopical administration to a vascular tissue include (1) coating orimpregnating a blood vessel tissue with a gel comprising an aptamer ofthe present invention, for delivery in vivo, e.g. by implanting thecoated or impregnated vessel in place of a damaged or diseased vesseltissue segment that was removed or by-passed; (2) delivery via acatheter to a vessel in which delivery is desired; and (3) pumping anaptamer composition of the present invention into a vessel that is to beimplanted into a patient.

The therapeutic compositions comprising an RNA aptamer polypeptide ofthe present invention are conventionally administered intravenously, asby injection of a unit dose, for example. The term “unit dose” when usedin reference to a therapeutic composition of the present inventionrefers to physically discrete units suitable as unitary dosage for thesubject, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required diluent; i.e., carrier or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with an RNA aptamer as described herein,dissolved or dispersed therein as an active ingredient. In a preferredembodiment, the therapeutic composition is not immunogenic whenadministered to a subject for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a patient without the production ofundesirable physiological effects such as nausea, dizziness, gastricupset and the like.

Pharmaceutically useful compositions comprising an RNA aptamer of thepresent invention can be formulated according to known methods such asby the admixture of a pharmaceutically acceptable carrier. Examples ofsuch carriers and methods of formulation can be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the aptamer. Such compositions cancontain admixtures of more than one aptamer.

Therapeutic or prophylactic compositions of the invention areadministered to an individual in amounts sufficient to modulatecoagulation and/or to treat or prevent cardiovascular disease. Theeffective amount can vary according to a variety of factors such as theindividual's condition, weight, sex and age. Other factors include themode of administration. Generally, the compositions will be administeredin dosages adjusted for body weight, e.g. dosages ranging from about 1μg/kg body weight to about 1 mg/kg body weight.

As noted above, the pharmaceutical compositions can be provided to theindividual by a variety of routes such orally, topically to a vasculartissue, intravenously, intraperitoneally, intramuscularly,subcutaneously, intra-cavity, transdermally, and can be delivered byperistaltic techniques. Representative, non-limiting approaches fortopical administration to a vascular tissue include (1) coating orimpregnating a blood vessel tissue with a gel comprising an aptamer ofthe present invention, for delivery in vivo, e.g. by implanting thecoated or impregnated vessel in place of a damaged or diseased vesseltissue segment that was removed or by-passed; (2) delivery via acatheter to a vessel in which delivery is desired; and (3) pumping anaptamer composition of the present invention into a vessel that is to beimplanted into a patient. Alternatively, the aptamer can be introducedinto cells by microinjection, or by liposome encapsulation.Advantageously, aptamers of the present invention can be administered ina single daily dose, or the total daily dosage can be administered inseveral divided doses.

Aptamers can be particularly useful for the treatment of diseases whereit is beneficial to inhibit coagulation, E2F activity, and/orangiogenesis factor activity (e.g. Ang1 or Ang2 activity), or preventsuch activity from occurring. The pharmaceutical compositions areadministered in therapeutically effective amounts, that is, in amountssufficient to generate a coagulation-, E2F activity-, and/orangiogenesis factor activity (e.g. Ang1 or Ang2 activity)-modulatingresponse, or in prophylactically effective amounts, that is in amountssufficient to prevent a coagulation factor from acting in a coagulationcascade, to prevent an E2F activity-mediated response, or to prevent anangiogenesis factor activity (e.g. Ang1 or Ang2 activity)-mediatedresponse. The therapeutically effective amount and prophylacticallyeffective amount can vary according to the type of aptamer. Thepharmaceutical composition can be administered in single or multipledoses.

Aptamers synthesized or identified according to the methods disclosedherein can be used alone at appropriate dosages defined by routinetesting in order to obtain optimal modulation of coagulation, E2Factivity, and/or angiogenesis factor activity (e.g. Ang1 or Ang2activity) while minimizing any potential toxicity. In addition,co-administration or sequential administration of other agents can bedesirable. For combination treatment with more than one active agent,where the active agents are in separate dosage formulations, the activeagents can be administered concurrently, or they each can beadministered at separately staggered times.

The dosage regimen utilizing the aptamers of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the patient; and the particularaptamer employed. A physician of ordinary skill can readily determineand prescribe the effective amount of the aptamer required to prevent,counter or arrest the progress of the condition. Optimal precision inachieving concentrations of aptamer within the range that yieldsefficacy without toxicity requires a regimen based on the kinetics ofthe aptamer's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of theaptamer.

In the methods of the present invention, the aptamers herein describedin detail can form the active ingredient, and are typically administeredin admixture with suitable pharmaceutical diluents, excipients orcarriers (collectively referred to herein as “carrier” materials)suitably selected with respect to the intended form of administration,that is, oral tablets, capsules, elixirs, syrup, suppositories, gels andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders includewithout limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents that can be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations that generally containsuitable preservatives are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl propionate, and the like, to form,e.g., alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions, and shampoos in cream or gel formulations.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines.

The compounds of the present invention can also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxy-ethylaspartamidephenol, orpolyethyl-eneoxidepolylysine substituted with palmitoyl residues.Furthermore, the compounds of the present invention can be coupled(preferably via a covalent linkage) to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polyethylene glycol (PEG), polylactic acid, polyepsilon caprolactone,polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels. Cholesterol and similar molecules can belinked to the aptamers to increase and prolong bioavailability.

V. METHODS OF IDENTIFYING APTAMERS

A method of identifying a ligand to a target from a candidate mixture ofpotential ligands is also provided in accordance with the presentinvention. Products, i.e. ligands, produced or identified by a method ofthe present invention are also provided.

In one embodiment the method preferably comprises: (a) preparing acandidate mixture of potential ligands; (b) contacting the candidatemixture with a target substrate in a lower stringency buffer, whereinligands having increased affinity to the target relative to thecandidate mixture bind to the target; (c) removing unbound candidatemixture; and (d) collecting the ligands that are bound to the target toproduce a first collected ligand mixture. More preferably, the methodfurther comprises: (e) contacting the first collected ligand mixturewith the target in a higher stringency buffer, wherein ligands havingincreased affinity to the target relative to the first collected ligandmixture bind to the target; (1) removing unbound ligands; and (g)collecting the ligands that are bound to the target to produce a secondcollected ligand mixture to thereby identify ligands to the target. Evenmore preferably, ligands in the first or second collected ligand mixtureare enriched or expanded by any suitable technique, e.g. amplification,prior to contacting the first collected ligand mixture with the targetin the higher stringency buffer, after collecting the ligands that boundthe target in the higher stringency buffer, or both. Optionally, thecontacting and expanding or enriching steps are repeated as necessary toproduce a desired ligand.

In an alternative embodiment, the method comprises: (a) immobilizing atarget on a substrate; (b) preparing a candidate mixture of potentialligands; (c) passing the candidate mixture over the substrate in a lowerstringency buffer, wherein ligands having increased affinity to theimmobilized target relative to the candidate mixture bind to theimmobilized target; (d) passing a wash buffer over the substrate toremove unbound candidate mixture; (e) passing an eluting buffer over thesubstrate to elute the ligands that are bound to the immobilized targetto produce an eluted ligand mixture; (f) passing the eluted ligandmixture over a substrate comprising immobilized target in a higherstringency buffer, wherein ligands having increased affinity to theimmobilized target relative to the eluted ligand mixture bind to theimmobilized target; (g) passing a wash buffer over the substrate toremove unbound ligands; and (h) passing an eluting buffer over thesubstrate to elute the ligands that are bound to the immobilized targetto thereby identify ligands to the target. With respect to thealternative embodiment, it also preferred that ligands in the elutedligand mixture are enriched or expanded by any suitable technique, e.g.amplification, prior to contacting the eluted ligand mixture with thetarget in the higher stringency buffer, after eluting ligands that boundthe target in the higher stringency buffer, or both. Optionally, thecontacting and expanding or enriching steps are repeated as necessary toproduce as desired ligand. Thus, it is possible that the secondcollected ligand mixture can comprise a single ligand.

The method is applicable to any target as defined herein for which aligand is sought. Representative targets are also disclosed in U.S. Pat.Nos. 5,756,291 and 5,817,785 herein incorporated by reference. In apreferred embodiment, the method further comprises amplifying the elutedligand to yield a ligand-enriched mixture, whereby a ligand to thetarget is identified. The ligand mixture can comprise a candidatemixture of any ligand as defined herein, including but not limited tonucleic acids. In this case, the candidate mixture of nucleic acidscomprises single strand nucleic acids. The single stranded nucleic acidscan comprise deoxyribonucleic acids.

Preferably, the single stranded nucleic acids are ribonucleic acids. Inthis case amplification can be accomplished, for example, via reversetranscriptase PCR reactions, as disclosed in U.S. Pat. No. 5,817,785,herein incorporated by reference. Optionally, the candidate mixture ofnucleic acids can comprise 2′-modified ribonucleic acids. For example,the 2′-modified ribonucleic acids can comprise 2′-fluoro (2′-F) modifiednucleic acids.

Representative wash and eluting buffers are disclosed in U.S. Pat. Nos.5,475,096 and 5,861,254, each of which incorporated herein by reference.In a preferred embodiment of the present invention in which a testnucleic acid mixture is incubated with target protein, the nucleicacid/protein mixture is filtered through a nitrocellulose filter andwashed with appropriate buffer to remove free nucleic acids.Protein/nucleic acids often remain bound to the filter. Filter washingprocedure can be optimized to reduce background binding. Suchoptimization of the filter washing procedures is within the skill of theordinary artisan.

Any suitable eluting buffer as would be apparent to one of ordinaryskill in the art after reviewing the disclosure of the present inventionpresented herein can also be employed in the present inventive method.For example, with respect to nucleic acid ligands, in order to proceedto the amplification step, selected nucleic acids must be released fromthe target after partitioning. This process is preferably done withoutchemical degradation of the selected nucleic acids and preferablyresults in amplifiable nucleic acids. For example, selected RNAmolecules can be eluted from nitrocellulose filters using a freshly madesolution containing 200 μl of a 7M urea, 20 mM sodium citrate (pH 5.0),1 mM EDTA solution combined with 500 μl of phenol (equilibrated with0.1M sodium acetate pH 5.2). A solution of 200 μl 7M urea with 500 μl ofphenol can also be employed. In this case, the eluted solution ofselected RNA can then extracted with ether, ethanol precipitated, andthe precipitate re-suspended in water. A number of different bufferconditions for elution of selected RNA from the filters can be used. Forexample, without limitation non-detergent aqueous protein denaturingagents such as quanidinium chloride, quanidinium thiocyanate, etc., asare known in the art, can be used. The specific solution used forelution of nucleic acids from the filter can be routinely selected byone of ordinary skill in the art.

As is understood in the art, the concentration of various ions, inparticular, the ionic strength, and the pH value impact on the value ofthe dissociation constant of the target/ligand complex. Thus, the terms“lower stringency” and “higher stringency” pertain to such bufferconditions (e.g. binding buffer conditions) as salt concentration, ionicstrength generally, pH, temperature, or organic solvents, as will bereadily appreciated by those skilled in the art after review of thedisclosure presented herein.

The method can further comprise step (i): repeating steps (e), (f) and(g) (preferred embodiment) or repeating steps (f), (g) and (h)(alternative embodiment) in a higher stringency buffer. Optionally, thehigher stringency buffer comprises a physiological buffer. As usedherein, “physiological conditions” means the salt concentration andionic strength in an aqueous solution that characterize fluids found inhuman metabolism commonly referred to as physiological buffer orphysiological saline. In general, these are represented by anintracellular pH of 7.1 and salt concentrations (in mM) of Na⁺: 3-15;K⁺: 140; Mg⁺²: 6.3; Ca⁺²: 10-14; Cl⁻: 3-15, and an extracellular pH of7.4 and salt concentrations (in mM) of Na⁺: 145; K⁺: 3; Mg⁺²: 1-2; Ca⁺²:1-2; and Cl⁻: 110. The use of physiological conditions in the ligandselection method is important, particularly with respect to thoseligands that can be intended for therapeutic use.

V.A. Modified SELEX

Systematic Evolution of Ligands by EXponential Enrichment, SELEX, isessentially a powerful iterative affinity purification process that canbe employed to isolate rare ligands from nucleic-acid combinatoriallibraries. As such, it is necessary to establish conditions under whichthe desired activity (e.g., binding a coagulation factor, angiogenesisfactor or E2F) can be detected in the initial randomized library priorto initiating the SELEX process. In addition, to purify ligandspossessing this activity from other sequences within the randomizedpool, the signal of the desired activity must be above the “noise” oflibrary binding to the partitioning scheme (i.e., the fraction ofsequences binding to the partitioning media due to target proteinbinding must be greater than those binding in a target independentmanner).

Generally, the SELEX process can be defined by the following series ofsteps:

(1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below; (b) to facilitate mimicry ofa sequence known to bind to the target; or (c) to enhance theconcentration of a given structural arrangement of the nucleic acids inthe candidate mixture. The randomized sequences can be totallyrandomized (i.e., the probability of finding a base at any positionbeing one in four) or only partially randomized (e.g., the probabilityof finding a base at any location can be selected at any level between 0and 100 percent).

(2) The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthe nucleic acids having the strongest affinity for the target.

(3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

(4) Those nucleic acids selected during partitioning as having therelatively higher affinity to the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

(5) By repeating the partitioning and amplifying steps above, the newlyformed candidate mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the nucleic acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield acandidate mixture containing one or a small number of unique nucleicacids representing those nucleic acids from the original candidatemixture having the highest affinity to the target molecule.

The desired characteristics for a given nucleic acid ligand can vary.All nucleic acid ligands are capable of forming a complex with thetarget. In some cases, it is desired that the nucleic acid ligand willserve to inhibit one or more of the biological activities of the target.In other cases, it is desired that the nucleic acid ligand serves tomodify one or more of the biological activities of the target. In othercases, the nucleic acid ligand serves to identify the presence of thetarget, and its effect on the biological activity of the target isirrelevant.

In order to identify conditions that are preferred for the initiation ofthe SELEX process (i.e., signal to noise ratio >2) a matrix strategy totest for target dependent versus target independent binding of thelibrary under different conditions is provided in accordance with thepresent invention. While this strategy is employed to identify SELEXconditions that are physiologic or that approach physiologic for thetarget protein, it could readily be applied to identify verynon-physiologic conditions if one desired to use an aptamer indownstream processes that require solvents that are non-physiologic.

In one embodiment of the matrix, the monovalent salt concentration andpH of the binding buffer are varied in combination to test monovalentsalt concentrations from 10 (low stringency) to 150 mM (high stringency)and a pH range from 7.0-8.0 in binding reactions with radiolabeledrandomized library RNA and varying concentrations of target protein. Allbinding buffers also contain physiologic concentrations of theappropriate divalent metal ions (depends on the in vivo compartment ofthe target) and excipient as needed to maintain the protein in solutionin a native state. This systematic variation of these criticalparameters allows for the rapid identification of the buffer conditionsand target concentration under which the SELEX process can besuccessfully initiated. In some cases, the initial conditions arenon-physiologic with respect to the monovalent salt concentration, thepH, or both. In these cases, the matrix strategy can be employed againin subsequent rounds of the SELEX process to determine when theconditions (i.e. binding buffer) of the SELEX can be changed. Thisstrategy is repeated every few rounds until the SELEX process can becontinued under physiologic conditions.

V.B. Toggle Method

Another embodiment of a method of identifying a ligand to a target froma candidate mixture of potential ligands is provided in accordance withthe present invention. Products, i.e., ligands, produced or identifiedvia the method are also provided in accordance with the presentinvention.

The method preferably comprises: (a) providing a target selected from afirst species of organism; (b) preparing a candidate mixture ofpotential ligands; (c) contacting the candidate mixture with the target,wherein ligands having increased affinity to the target from the firstspecies of organism relative to the candidate mixture bind to theimmobilized target from the first species of organism; (d) removingunbound candidate mixture; (e) collecting the ligands that are bound tothe target from the first species of organism to produce a firstcollected ligand mixture for the target; (f) contacting the firstcollected ligand mixture with a target from a second species oforganism, the target from the second species having at least a portionthereof that is substantially homologous to the same portion in thetarget from the first species, wherein ligands having increased affinityto the target from the second species relative to the first collectedligand mixture bind to the target; (g) removing unbound first collectedligand mixture; and (h) collecting the ligands that are bound to thetarget from the second species of organism to form a second collectedligand mixture thereby identify ligands to the target.

Preferably, ligands in the first or second collected ligand mixture areenriched or expanded by any suitable technique, e.g. amplification,prior to contacting the first collected ligand mixture with the targetfrom the second species of organism, after collecting the ligands thatbound the target from the second species of organism, or both.Optionally, the contacting and expanding or enriching steps are repeatedas necessary to produce a desired ligand. Thus, it is possible that thesecond collected ligand mixture can comprise a single ligand.

Another embodiment of a method of identifying a ligand to a target froma candidate mixture of potential ligands in accordance with the presentinvention comprises: (a) immobilizing a target on a substrate, thetarget comprising a target selected from a first species of organism;(b) preparing a candidate mixture of potential ligands; (c) passing thecandidate mixture over the substrate, wherein ligands having increasedaffinity to the immobilized target from the first species of organismrelative to the candidate mixture bind to the immobilized target fromthe first species of organism; (d) passing a wash buffer over thesubstrate to remove unbound candidate mixtures; (e) passing an elutingbuffer over the substrate to elute the ligands that are bound to theimmobilized target from the first species of organism to produce aneluted ligand mixture; (f) passing the eluted ligand mixture over asubstrate comprising an immobilized target from a second species oforganism, the target from the second animal species contacting the firstcollected ligand mixture, the target from the second species having atleast a portion thereof that is substantially homologous to the sameportion in the target from the first species, wherein ligands havingincreased affinity to the target from the second species relative to thefirst collected ligand mixture bind to the immobilized target; (g)passing a wash buffer over the substrate to remove unbound eluted ligandmixture; and (h) passing an eluting buffer over the substrate to elutethe ligands that are bound to the immobilized target from the secondspecies of organism to thereby identify ligands to the target.

Preferably, ligands in the first or second collected ligand mixture areenriched or expanded by any suitable technique, e.g. amplification,prior to contacting the first collected ligand mixture with the targetfrom the second species of organism, after collecting the ligands thatbound the target from the second species of organism, or both.Optionally, the contacting and expanding or enriching steps are repeatedas necessary to produce a desired ligand. Thus, it is possible that thesecond collected ligand mixture can comprise a single ligand.

Optionally, the method can further comprise further comprising step (i):repeating steps (f), (g) and (h) one or more times, wherein eachadditional time alternates between the target from the first species andthe target from the second species. The method can also further compriseamplifying the eluted ligand to yield a ligand-enriched mixture, wherebya ligand to the target is identified.

The method is applicable to any target and to any ligand as definedherein for which a ligand is sought. Representative targets are alsodisclosed in U.S. Pat. No. 5,756,291, herein incorporated by reference.Alternation of the target between homologous proteins of differentspecies ensures that the products of selection will bind to bothproteins, most likely to domains conserved between the two proteins.Because conserved domains tend to be functionally important, driving theselection with homologous proteins can be advantageous even when speciescross-reactivity is not necessarily sought. Similarly, the toggleapproach is generalizable to homologous proteins of different viralstrains and to related proteins of the same species (such as receptor orligand families with redundant or overlapping function), wherecross-reactivity can improve in vivo efficacy. Thus, the phrase “thetarget from the second species having at least a portion thereof that issubstantially homologous to the same portion in the target from thefirst species” includes but is not limited to conserved domains.

The term “substantially homologous” in the context of two or morepolypeptide sequences is measured by polypeptide sequences having about35%, or 45%, or preferably from 45-55%, or more preferably 55-65%, ormost preferably 65% or greater amino acids which are identical orfunctionally equivalent. Percent “identity” and methods for determiningidentity are defined herein below. Further, this term also encompassesproteins typically referred to in the art as homologues, e.g. porcineand human thrombin as disclosed in the Examples.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv Appl Math2:482, by the homology alignment algorithm of Needleman and Wunsch(1970) J Mol Biol 48:443, by the search for similarity method of Pearsonand Lipman (1988) Proc Natl Acad Sci USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group,Madison, Wis.), or by visual inspection (See generally, Ausubel et al.(1992)).

A preferred algorithm for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al. (1990) J Mol Biol 215: 403-410. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold. These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength W=11, an expectationE=10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. SeeHenikoff and Henikoff (1989) Proc Natl Acad Sci USA 89:10915.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. See, e.g., Karlin and Altschul (1993) Proc Natl Acad SciUSA 90:5873-5887. One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

In a preferred embodiment, the method further comprises amplifying theeluted ligand to yield a ligand-enriched mixture, whereby a ligand tothe target is identified. The ligand mixture can comprise a candidatemixture of nucleic acids. In this case, the candidate mixture of nucleicacids can comprise single strand nucleic acids. The single strandednucleic acids can comprise deoxyribonucleic acids.

Preferably, the single stranded nucleic acids are ribonucleic acids. Inthis case amplification can be accomplished, for example, via reversetranscriptase PCR reactions, as disclosed in U.S. Pat. No. 5,817,785,herein incorporated by reference. Even more preferably, the candidatemixture of nucleic acids comprises 2′-modified ribonucleic acids. Evenmore preferably, the 2′-modified ribonucleic acids comprise 2′-fluoro(2′-F) modified nucleic acids.

Contemplated species of organism include plants, animals, bacteria,fungi, viruses or other organism. Preferred species of animals includewarm-blooded vertebrates, including but not limited to humans, rats,mice, pigs, apes, monkeys, cats, dogs, cattle, oxen, sheep, goats andhorses, or other mammal typically used in experiments. Additionalspecies include domesticated fowl, i.e., poultry, such as turkeys,chickens, ducks, geese, guinea fowl, and the like, as they are also ofeconomical importance to humans. Thus, the term “avian” as used hereinrefers to any avian species, including but not limited to Gallinaceasp., chicken, turkey, duck, goose, quail and pheasant. Chicken iscurrently preferred.

Fish represent a category of animals of interest for agricultural andecological reasons. Representative fish species include, but are notlimited to, trout, salmon, carp, shark, ray, flounder, sole, tilapia,medaka, goldfish, guppy, molly, platyfish, swordtail, zebrafish, loach,catfish, and the like. Representative general techniques that areapplicable to fish have been described by Ozato et al, Cell Differ.,19:237-244 (1986), Inoue et al, Cell Differ. Dev., 29:123-128 (1990),Rokkones et al, J. Comp. Physiol. B, 158:751-758 (1989), and Guyomard etal, Biochimie, 71:857-863 (1989), describing preparation of transgenicmedaka, medaka, salmon and trout, respectively.

LABORATORY EXAMPLES

The following Laboratory Examples have been included to illustratepreferred modes of the invention. Certain aspects of the followingLaboratory Examples are described in terms of techniques and proceduresfound or contemplated by the present inventors to work well in thepractice of the invention. These Laboratory Examples are exemplifiedthrough the use of standard laboratory practices of the inventors. Inlight of the present disclosure and the general level of skill in theart, those of skill will appreciate that the following examples areintended to be exemplary only and that numerous changes, modificationsand alterations can be employed without departing from the spirit andscope of the invention.

Example 1 Initial Condition Matrix to Coagulation Factor VIIa

Referring now to FIG. 8, radiolabeled 2′fluoropyrimidine-modifiedlibrary RNA was incubated with varying concentrations of coagulationfactor VIIa in 9 different buffers, and target-bound versus free RNA wasdetermined using the double-filter nitrocellulose filter binding assay(Wong and Lohman, 1993, Proc. Natl. Acad. Sci. USA 90, 5428-5432). Forall conditions, the binding buffers contain 20 mM Hepes at the pH shown,2 mM CaCl₂, and NaCl at the concentration shown. From the left to rightin each set of 4, the factor VIIa concentration is 5 μM, 1.67 μM, 0.56μM and no target. The fraction of the pool that binds factor VIIadecreases dramatically as the monovalent salt increases, but isrelatively unaffected by the change of pH at a given NaCl concentration.In addition, the fraction of RNA bound in the absence of target is muchgreater at lower NaCl concentrations and lower pH.

In this Example, there is essentially no target binding above backgroundunder physiologic conditions (pH 7.4, 150 mM NaCl). Thus, if the SELEXprocess were initiated under these conditions it would fail outright.Likewise, at pH 7.0, 50 mM NaCl, there is significant target dependentbinding, but also significant target independent binding. Were the SELEXinitiated under these conditions, it would also likely fail. Preferredinitiating conditions are pH 7.4, 50 mM NaCl, with a relatively high(μM) concentration of factor VIIa.

Example 2 Initial Condition Matrix to Coagulation Factor IXa

Referring now to FIG. 9, radiolabeled 2′fluoropyrimidine-modifiedlibrary RNA was incubated with varying concentrations of coagulationfactor IXa in 6 different buffers, and target-bound versus free RNA wasdetermined using the double-filter nitrocellulose filter binding assay.For all conditions, the binding buffers contain 20 mM Hepes at the pHshown, 2 mM CaCl₂, and NaCl at the concentration shown. From left toright in sets of 6, the FIXa concentration decreases 2 fold at eachpoint from 5 μM to 0.31 μM, and the sixth point is the no targetcontrol.

The fraction of the pool that binds factor IXa decreases dramatically asthe NaCl concentration increases. In this Example, there is low butdetectable binding of the pool to factor IXa under physiologicconditions, and the SELEX process could be successfully initiated underthese conditions. However, a preferred initiating condition would be pH7.4, 50 mM NaCl, followed by a change to physiologic buffer in the earlyrounds of the SELEX process.

Example 3 Toggle SELEX

Species cross-reactivity facilitates the pre-clinical evaluation ofpotentially therapeutic molecules in animal models. This Exampledescribes an in vitro selection strategy in which RNA ligands (aptamers)that bind both human and porcine thrombin were selected by “toggling”the protein target between human and porcine thrombin during alternatingrounds of selection. The “toggle” selection process yielded a family ofaptamers, all of which bound both human and porcine thrombin with highaffinity. TOGGLE-25, a characteristic member, inhibited two ofthrombin's most important functions: plasma clot formation and plateletactivation. If appropriate targets are available, the toggle strategy isa simple measure that promotes cross-reactivity and can be generalizableto related proteins of the same species as well as to othercombinatorial library screening strategies. This strategy shouldfacilitate the isolation of ligands with needed properties for genetherapy and other therapeutic and diagnostic applications.

Anti-thrombin therapeutics have been of great interest due to thrombin'scentral role in blood coagulation as well as to its suggested role incellular proliferation (intimal hyperplasia) following vascular injury.To develop a method for the generation of aptamers that would beclinically useful in humans and testable in animal models, a novelselection strategy was applied to this previously validated target.Nuclease-resistant RNA ligands that bind both human and porcine thrombinwere selected by “toggling” the protein target between human and porcinethrombin during alternating rounds of selection (FIG. 31). Suchcross-reactive aptamers inhibit both porcine and human thrombin activityas might be expected from ligands made to bind evolutionally conservedregions of a protein.

A library containing approximately 10¹⁴ different RNAs was screened forthose molecules that bind both human and porcine thrombin. In the firstround of in vitro selection, the starting library was incubated withboth human and porcine thrombin (F2). RNAs that bound either proteinwere recovered and amplified to generate a library enriched inF2-binding RNAs. In round 2 of selection, the enriched library of RNAswas incubated with human F2 alone, and bound RNAs were recovered andamplified to generate a library of RNAs that had been further enrichedfor members that bound surfaces on human F2 (FIG. 31). In round 3, thishuman-focused library was incubated with porcine F2, and the subset ofRNAs that bound the porcine protein were recovered and amplified togenerate a library that had been further enriched for binding structuralmotifs that were conserved between the F2 homologs. This “toggle”selection process was repeated during rounds 4-13 of SELEX utilizing thehuman protein for even rounds and the porcine protein for odd rounds. Inparallel, thirteen (13) rounds of standard SELEX were performed againstthe individual human and porcine proteins. To determine if toggle SELEXenriched for aptamers that cross-reacted with both human and porcine F2,binding properties of the RNA pools from various rounds of selectionwere evaluated.

Early in the selection (round 3), no significant differences in thebinding affinities of RNA pools from the human, toggle, or porcineselections were observed for either human F2 (FIG. 32A) or porcine F2(FIG. 32B). By round 11, however, RNA pools selected against human F2alone had less avid binding for porcine F2 (FIG. 32C), and RNA poolsselected against porcine F2 alone had less avid binding for human F2(FIG. 32D), suggesting the presence of species-specific ligands withinthese pools; whereas RNA pools generated via toggle SELEX bound bothhuman and porcine F2 as well as did RNA pools generated against eachprotein alone (FIGS. 32C and 32D). With continued selection, thedifferential binding of porcine selection RNA pools for human F2 wasdiminished (FIG. 32E), and the differential binding of the humanselection RNA pools for porcine F2 was lost (FIG. 32F), indicating theloss of species-specific ligands from these pools.

Three in vitro selections were performed in parallel: human thrombin,porcine thrombin, and a toggle selection using alternating rounds ofhuman and porcine thrombin in a SELEX process as disclosed in Examples 1and 2 above. After thirteen rounds of the SELEX process, the pools werecloned and sequenced. The random sequences of seven clones of highaffinity for one another are shown in FIG. 11. Some of the sequencesshown in FIG. 11 were represented in more than one selection but areonly listed once in FIG. 11 for clarity.

Thus, the amplification products from round 13 of each selection werecloned and sequenced. Thirteen clones analyzed from the human selectionhad identical sequences (HUMAN-1). Consistent with round 13 binding data(FIGS. 32E and 32F), HUMAN-1 RNA bound both human and porcine F2 withhigh affinity. Thus, the selection to human F2 appears to have beendriven to completion and yielded an aptamer that binds an epitopepresent on both the human and porcine proteins. Sequences from earlierrounds, when multiple aptamers were still present, were not analyzed.All sequences from the toggle selection and 10 of 14 sequences from theporcine selection shared a conserved motif with the predominant humanselection sequence: ACAAAGCUGRAGWACUUA (SEQ ID NO:227), where Rrepresents A or G and W represents A or U. RNAs possessing the consensussequence shared similarly high binding affinities for both human F2(K_(d)'s ranging from 1 to 4 nM) and porcine F2 (all K_(d)'s<1 mM). Arepresentative RNA, TOGGLE-25, bound human F2 with a K_(d) of 2.8±0.7 nMand bound porcine F2 with a K_(d) of 83±3 pM. A unique RNA from theporcine selection (PIG-10) bound porcine F2 with a K_(d) of 50±8 pM butbound human F2 with a K_(d) of greater than 600 nM, demonstratinggreater than 10,000-fold specificity for porcine F2 over human F2. Thus,the porcine thrombin selection yielded two classes of high affinityligands. One recognizes an epitope that is conserved on human F2 whilethe other recognizes an epitope that is not conserved. Interestingly,the two RNAs compete for binding to porcine F2, suggesting that theybind overlapping regions of the protein. These two RNAs are furthercharacterized below.

A fundamental problem in the development of new therapies is thatpotentially useful therapeutic agents might not progress from bench topto clinical trials due to lack of demonstrated efficacy in animalmodels. Drug discovery strategies generally employ human targets, andthe inability to cross-react with homologous targets of other speciescan interfere with pre-clinical testing. This Example discloses ahigh-affinity inhibitor of the protease thrombin using a novel in vitroselection strategy. The “toggle” selection strategy exploits themalleability of iterative in vitro techniques and is generalizable toother combinatorial library screening strategies. Alternation of thetarget between homologous proteins of different species ensures that theproducts of selection will bind to both proteins, most likely to domainsconserved between the two proteins. Because conserved domains tend to befunctionally important, driving the selection with homologous proteinscan be advantageous even when species cross-reactivity is notnecessarily sought. Similarly, the toggle approach is generalizable tohomologous proteins of different viral strains and to related proteinsof the same species (such as receptor or ligand families with redundantor overlapping function), where cross-reactivity can improve in vivoefficacy.

For a relatively well-conserved protein like thrombin (85% amino acidhomology between human and bovine thrombin (Butkowski, R. J., et al.(1977) J. Biol. Chem. 252:4942-4957), aptamers selected against humanthrombin might cross-react with thrombin of other species. Asingle-stranded DNA aptamer to human thrombin inhibits thrombin functionin canine and ovine models, although relative binding affinities havenot been reported. DeAnda, A., Jr., et al. (1994) Ann. Thorac. Surg.58:344-350; Griffin, L. C., et al. (1993) Blood 81:3271-3276. The singleaptamer identified from round 13 of the selection against human thrombinhappened to cross-react with porcine thrombin; however, aptamers fromearlier rounds where species-specificity was observed were not analyzed,and these RNA pools likely included species-specific aptamers. Theidentification of a species-specific aptamer from the porcine selectionunderscores the point that—even for selection against a well-conservedprotein—species cross-reactivity should not be assumed.

The “toggle” selection yielded a family of aptamers, all of which boundboth human and porcine thrombin with high affinity. Although selectionagainst both targets could theoretically sacrifice affinity for thehuman target, this “toggle” family demonstrated affinity for humanthrombin similar to RNAs generated against human thrombin alone.Furthermore, the potential loss in binding affinity should be outweighedby the potential gain in activity expected from driving selection towardevolutionally conserved regions of the protein. TOGGLE-25, acharacteristic member of the “toggle” family, inhibited two ofthrombin's most important functions: plasma clot formation and plateletactivation. Truncation of the full-length aptamer to approximately 40 ntor fewer is preferred for efficient chemical synthesis, but minimizationcan have varying effects on binding and inhibitory activity. A 25-nttruncate (TOGGLE-25t) retained the inhibitory activities of thefull-length aptamer and demonstrated greater potency, particularly inplasma-based assays. The superiority of the TOGGLE-25t truncate in theseassays might be attributable to slightly higher affinity for thrombin aswell as theoretically less nonspecific binding to positively-chargedplasma proteins.

Thrombin aptamers can fulfill a clinical need for more and betteranticoagulants and antithrombotics. Widely utilized agents such asheparin and platelet surface receptor antagonists are effective but arelimited by bleeding complications due to Heparin InducedThrombocytopenia and irreversibility, respectively. The plasma stabilityof our aptamer has been increased by the substitution ofendonuclease-resistant nucleotides, which typically confer an in vitrohalf-life of greater than 5 hours as compared to 30 to 60 minutes forDNA and seconds for unmodified RNA. Pieken, W. A., et al. (1991).Science 253:314-317; Beigelman, L., et al. (1995) J. Biol. Chem.270:25702-25708. Due to its small size and stability in plasma, its invivo half-life is governed by the rate of renal clearance, which can bereduced by the addition of various inert high molecular weight orhydrophobic groups Willis, M. C., et al. (1998) (published erratumappears in Bioconjug Chem 1998 September-October; 9(5):633) BioconjugChem. 9:573-582; Tucker, C. E., et al. (1999). Journal ofChromatography. B, Biomedical Sciences & Applications 732:203-212.Depending on the planned application, therefore, our aptamer can bemodified to have an in vivo half-life of minutes or hours. In addition,the high affinity of the aptamer can be exploited to more effectivelyblock the thrombogenic and mitogenic effects of thrombin on platelets,fibroblasts, endothelium, and smooth muscle, which are believed to bemediated by proteolytic activation of a novel family of receptors,Protease-Activated Receptors or PARs.

Aptamers are a promising class of molecules, for target validation aswell as for diagnosis and therapy. Nuclease-resistant aptamers havealready proven to be useful reagents for the study of extracellulartargets in a variety of disease processes, and animal studies have pavedthe way for the first clinical trials of an aptamer against VEGF in thewet form of age-related macular degeneration. In addition, genetherapeutic methods utilizing viral constructs have been used to“express” unmodified RNA aptamers against intracellular targets.Sullenger, B. A., et al. (1990) Cell 63:601-608; Good, P. D., et al.(1997) Gene Ther. 4:45-54. Species cross-reactivity will greatlyfacilitate the transition of these molecules from in vitro assays toanimal models to human subjects. If the appropriate targets areavailable, the toggle strategy is a simple measure that promotescross-reactivity. Because the selection process is performed in vitro,possible schemes to manipulate the specificity of an aptamer for thetargeted protein(s) are virtually unlimited.

Accordingly, the present invention provides a method to isolate thetherapeutic compounds against human (or other species) molecular targetssuch that the therapeutic compound cross-reacts with homologousmolecular targets from another species. For example, this process canentail the selection of a therapeutic compound against a human targetmolecule of interest followed by selection against a homologous moleculefrom another species. This process yields compounds that can beevaluated in a predetermined preclinical animal model by isolatingmolecules that cross-react with the species that is of interest.

As disclosed in this Example, an aptamer to human thrombin that crossreacts with human porcine thrombin has been produced using this method.Moreover, this toggle selection approach is expected to yield compoundsthat interact with functionally conserved regions of the target moleculethat are likely to be the functionally important regions on the target.

Example 4 Inhibition of the Activity of Coagulation Factor IXa

Referring now to FIG. 2A, it was shown that RNA 9.3 (SEQ ID NO:3)inhibits the FIXa/FVIIIa catalyzed activation of FX. In FIG. 2A, factorIXa (0.5 nM) was equilibrated with no RNA (▴), 10 nM control RNA (▪), or10 nM RNA 9.3 (). The FX activation reaction was initiated by theaddition of FVIIIa (1 nM), PC/PS vesicles (100 μM) and FX (200 nM). Theamount of FXa formed over time at 37° C. was measured.

Referring now to FIG. 2B, it was shown that RNA 9.3 (SEQ ID NO:3)prolongs the clotting time of human plasma. The clotting time of normalhuman plasma was measured in an aPTT assay in the absence of RNA(striped bar), or in the presence of increasing concentrations ofcontrol RNA (solid bar) or RNA 9.3 (SEQ ID NO:3) (open bar). Theclotting time is expressed as the mean ±sem for duplicate measurements.

Example 5 Truncated FIXa Aptamers

The primary sequence of the minimal FIXa aptamers 9.3^(t) and 9.20^(t)are set forth in Table 1 above. The apparent K_(d) for binding of the9.3 truncate 9.3^(t) to FIXa ranges from about 1 to about 3 nM, and toFIX ranges from about six (6) to about ten (10) nM. The apparent K_(d)of the 9.20^(t) truncate 9.20^(t) for FIXa is ˜100-200 nM.

Referring now to FIGS. 3A-3C, 9.3^(t) prolongs the clotting time of pigplasma as well as clotting time in vivo in a dose dependent manner asdetermined by aPTT clotting assays, ACT assays, and PT assays. The doseresponse curve is consistent with substantially complete inactivation ofFIX/FIXa activity.

Examples 6A and 6B Systemic Anticoagulation Model in Swine

Examples 6A and 6B pertain to the evaluation of the ability of thetruncated FIX/FIXa aptamer, RNA 9.3 truncated aptamer 9.3^(t) (SEQ IDNO:70), to systemically anti-coagulate small (2-4 kg) swine. Theevaluation included three groups with 3 animals per group. The groupsare vehicle control (20 mM Hepes, pH 7.4; 150 mM NaCl; 2 mM CaCl₂),about 0.5 mg/kg aptamer in vehicle, and about 1.0 mg/kg aptamer invehicle.

The following procedures were performed for each animal. Underanesthesia, a venous catheter was placed in the femoral vein of the pigfor sample injection, and an arterial catheter was placed in the femoralartery for serial withdrawal of blood samples. Prior to injection, andat times thereafter, blood samples were taken and processed. One portionof the sample (˜0.4 mls) was used to measure, on site, an activatedclotting time (ACT) to establish a baseline whole blood clot time foreach animal

A second portion of the sample (˜2.7 mls) was aliquoted intocitrate-containing Vacutainer™ tubes and processed to prepare plasma forcoagulation assays (e.g. aPTT's). A third portion of sample (˜2.5 mls)was aliquoted into EDTA-containing Vacutainer™ tubes and processed toprepare plasma to be used in the measurement of the plasma aptamerconcentration. In addition, CBC's were performed prior to injection andat the end of the study.

Example 6A Change in Clot Times Following Injection of Vehicle orFIX/FIXa Aptamer

Referring now to FIG. 3B, the change in the Activated Clotting Time(ACT) following treatment with aptamer was measured. The ACT increase isthe ratio of the (pre-injection ACT/post injection ACT) for each timepoint, 1.0=no change (time 0=pre-injection). There is a cleardose-dependent increase in the ACT of the aptamer but not vehicletreated animals, with a peak at 4 min post injection for the low dose,and 4-6 min post injection for the high dose. At the lower dose, theeffect persists out to nearly 15 minutes post injection, while at thehigher dose the ACT is still elevated at the conclusion of theexperiment. Data is presented as the mean ±sem.

Referring now to FIG. 3C, the change in the Prothrombin Time (PT) andactivated Partial Thromboplastin Time (aPTT) following treatment withaptamer was also measured. The increase in the PT or APTT is the ratioof the (pre-injection clot time/post injection clot time) for each timepoint, 1.0=no change (time 0=pre-injection). The PT is sensitive toinhibitors of coagulation factors II, V, VII, and X, but not toinhibitors of factors VIII or IX. The APTT is sensitive to inhibitors offactor IX/IXa. There is a clear dose-dependent increase in the aPTT ofaptamer treated but not vehicle treated animals, with the peak increaseoccurring at the earliest time point taken (1 minute). In contrast, thePT is unaffected by vehicle or either dose of aptamer, demonstrating thefunctional specificity of the FIX/FIXa aptamer for its target versusother coagulation factors.

An important difference between the aPTT/PT assays and the ACT assay isthat in the aPTT and PT assays, the plasma is pre-warmed (3-5 minutes)ex vivo prior to measuring the clotting time. Therefore, while the ACTlikely reflects the dynamics of the drug action in vivo (plasmadistribution, kinetics of target binding etc.), the aPTT likely reflectsthe total plasma concentration of the aptamer at a given timepost-injection. In FIG. 3C, data is presented as the mean ±sem.

Example 6B Change in Clot Times Following Injection of 0.5 mg/kg5′Cholesterol Modified FIX/FIXA Aptamer Versus Unmodified FIX/FIXaAptamer

Referring now to FIGS. 5A and 5B, the change in the ACT followingtreatment with the cholesterol-modified aptamer was evaluated. Theaddition of cholesterol to an aptamer is expected to increase the plasmaresidence time of an aptamer following intravenous injection. The ACTincrease is the ratio of the (pre-injection ACT/post injection ACT) foreach time point, 1.0=no change (time 0=pre-injection). The duration ofthe increase in the ACT of the pig is clearly longer following injectionof the cholesterol modified aptamer, RNA 9.3 truncated aptamer 9.3^(t-c)(SEQ ID NO:70)-C, versus RNA 9.3 truncated aptamer 9.3^(t) (SEQ IDNO:70). Following treatment with 9.3^(t), the effect persists out tobetween 15 to 20 minutes post injection, while following treatment withRNA 9.3 truncated aptamer 9.3^(t-c) (SEQ ID NO:70)-C, the ACT is stillelevated at the conclusion of the experiment (about 180 minutes). Datais presented in FIGS. 5A and 5B is the average of duplicate measurementsat each time point.

Referring now to FIG. 5C, the change in the PT and aPTT followingtreatment with cholesterol-modified aptamer is depicted. The increase inthe PT or aPTT is the ratio of the (pre-injection clot time/postinjection clot time) for each time point, 1.0=no change (time0=pre-injection). The PT is sensitive to inhibitors of coagulationfactors II, V, VII, and X, but not to inhibitors of factors VIII or IX.The aPTT is sensitive to inhibitors of factor IX/IXa. There is a cleardose-dependent increase in the aPTT of aptamer treated animals with thepeak increase occurring at the earliest time point taken (1 minute);whereas the PT is unaffected by either aptamer (see also FIG. 3C).

Continuing with FIG. 5C, following treatment with RNA 9.3 truncatedaptamer 9.3t (SEQ ID NO:70), the effect persists out to between about 30minutes post injection (see also FIG. 3C), while following treatmentwith RNA 9.3 truncated aptamer 9.3t-c (SEQ ID NO:70)-C, the aPTT isstill elevated at the conclusion of the experiment (about 180 minutes).In addition, although both aptamers give a similar initial aPTTincrease, the increase in the aPTT of the pig following injection of RNA9.3 truncated aptamer 9.3^(t-c) (SEQ ID NO:70)-C is greater than thatincrease following treatment with RNA 9.3 truncated aptamer 9.3^(t) (SEQID NO:70). This indicates that the plasma concentration of RNA 9.3truncated aptamer 9.3^(t) (SEQ ID NO:70)-C decreases more slowly thanthe plasma concentration of RNA 9.3 truncated aptamer 9.3^(t) (SEQ IDNO:70) following intravenous injection. Data is presented in FIG. 5C isthe average of duplicate measurements at each time point.

Example 7 Inhibition of the Activity of Coagulation Factor Xa

RNA 10.14 (SEQ ID NO:73) inhibits the FXa/FVa catalyzed activation ofprothrombin. As shown in FIG. 7A, factor Xa (0.5 nM) was equilibratedwith no RNA (▴), 100 nM control RNA (▪), or 100 nM RNA 10.14 (), theprothrombin activation reaction was initiated by the addition of FVa (1nM), PC/PS vesicles (100 μM) and prothrombin (200 nM), and the amount ofthrombin formed over time at 37° C. was measured.

Referring now to FIG. 7B, the clotting time of normal human plasma wasmeasured in a PT assay in the absence of RNA (striped bar), or in thepresence of increasing concentrations of control RNA (solid bar) or RNA10.14 (open bar). The clotting time is expressed as the mean ±sem forduplicate measurements.

Example 8 Inhibition of Platelet Activation Using Thrombin RNA Aptamers

In this Example the ability of a thrombin RNA aptamer produced by thetoggle SELEX method described in Example 3 to inhibit plateletactivation was evaluated. Referring to FIGS. 11 and 12, human plateletswere isolated and activated by 1 nM human thrombin, in the presence of30 nM toggle 25 (TOG 25, SEQ ID NO: 55) RNA aptamer ornitrocellulose-binding control RNA aptamer. The protease activatedreceptor-1 (PAR-1) peptide ligand, SFLLRN (SEQ ID NO: 72), was used as apositive control. As shown in FIG. 12, the TOG 25 aptamer inhibitedplatelet activation to about 10%, which compared favorably to theplatelet samples that included no activator.

Referring now to FIG. 13, porcine platelets were isolated and activatedby 2 nM porcine thrombin in the presence of the 20 nM TOG 25 RNA aptameror nitrocellulose-binding control RNA aptamer. As shown in FIG. 13, theTOG 25 aptamer was also able to inhibit pig thrombin in that theactivation of the pig platelets was maintained at levels compared tothat observed in samples where thrombin was not present.

Referring now to FIG. 14, activated partial thromboplastin time (aPTT)assays were performed on human and pig plasma in the presence ofincreasing concentrations of the toggle 30 (TOG 30, SEQ ID NO: 56) RNAaptamer that was also prepared using the toggle SELEX method describedin Example 3. As shown in FIG. 14, comparable effects were observed inboth human and pig plasma with the TOG 30 RNA aptamer. A control aptamerhad no effect on aPTT at these concentrations.

The data presented in this Example and in FIGS. 11-14 clearly indicatethat the toggle SELEX method disclosed herein to provide aptamers thatexhibit selectivity for multiple species. The toggle SELEX method of thepresent invention thus provides a methodology wherein an aptamer that isselected for a test species as well as an ultimate species in which thetreatment is to be applied (e.g., pigs and humans) can be identified.This compatibility facilitates the identification of aptamers or otherligands that have activity in a test animal will also have activity in asubject that will ultimately be treated with the aptamer or otherligand.

Example 9 Mutation Analysis of Antithrombin RNA Aptamer

A proposed secondary structure of a 25 mer truncate of TOG 25 (TOG 25short, SEQ ID NO: 57), is shown in FIG. 15. The nonmutated or“wild-type” truncate binds human thrombin with a K_(d) of approximately1 nM. Continuing with FIG. 15, the binding affinity of various mutantsdesigned to disrupt the proposed stem region, the BULGE region, and theLOOP sequences are shown and are summarized as follows. In the NO STEMmutant, wherein cytosines are mutated to guanidine, the binding affinityis approximately 1 micromolar. In the BULGEUs region, uracil moietiesare mutated to adenine and the resulting dissociation constant is muchgreater than 1 nM. These mutations thus substantially impair the bindingaffinity of the TOG 25 short aptamer to thrombin. In the LOOPUImutation, uracil to adenine, produces an aptamer having a dissociationconstant of about 250 nM. The LOOPU2 mutation, also uracil to adenine,produces an aptamer having a dissociation constant of about 1 nM. Thus,the mutations in the STEM and BULGE regions have greater impact thanthose in the LOOP regions.

Referring now to FIG. 16, the ability of the full-length TOG 25 RNAaptamer, the TOG 25 short RNA aptamer, and the BULGEUs RNA aptamer toinhibit platelet activation is analyzed. Human platelets were isolatedand activated by 1 nM human thrombin, in the presence of increasingcombination of full length TOG 25 (TOG 25 FL, SEQ ID NO: 55), truncatedTOG 25 (TOG 25 short, SEQ ID NO: 57), or a non-binding human truncate(BULGEUs, SEQ ID NO: 59). Both the TOG 25 FL and TOG 25 short aptamerssubstantially inhibit platelet activation, with observed levels ofplatelet activation below 20%. In contrast, the mutant BULGEUs aptamerdid not demonstrate an ability to inhibit platelet activation, withactivation levels of approximately 90%. These data thus provide guidanceto the design and selection of aptamers that provide the desiredmodulator effects.

Example 10 Inhibition of the Activity of Coagulation Factor VIIa

Referring now to FIGS. 19A-19B, a factor VIIa aptamer RNA 16.3 (SEQ IDNO: 41) inhibits the activity of coagulation factor VIIa. RNA 16.3inhibits the VIIa/TF catalyzed activation of FX. Factors VIIa and X wereequilibrated with no RNA (□) 1 mM mutated non-binding RNA 16.3 m4 (▪) asa composition control, or 1 μM RNA 16.3 (▴), and the FX activation wasinitiated by the addition of lapidated tissue factor. The amount of FXaformed over time at 25° C. was measured. The amount of FXa formed overtime is expressed as the mean sem for three or more experiments.

In FIG. 19B, it is shown that RNA 16.3 prolongs the tissue factorinduced clotting time of human plasma. The clotting time of human plasmawas measured in a PT assay in the absence of RNA (striped bar), whereinthe presence of varying concentrations of RNA 16.3 mutated non-bindingM4 (solid bar) or 16.3 (open bar). The clotting time is expressed as themean ±sem for duplicate experiments.

Example 11 Inhibition of the Activity of Coagulation Factor VIIa

A nuclease-resistant 2′amino pyrimidine-modified RNA aptamer that canblock tissue factor/FVIIa catalyzed FX activation, and that can prolongthe clotting time of human plasma are disclosed herein above in SectionII.B., in FIG. 10, and in Example 10. While a potent antagonist at roomtemperature, it was discovered that this antagonist is much less potentat physiologic temperature (likely due to the reduced stability ofduplexes containing the 2′amino modification). Therefore, this Examplepertains to the generations of additional FVIIa antagonists byperforming SELEX using a 2′fluoropyrimidine-modified RNA library at 37°C. (this modification yields duplexes with enhanced stability comparedto standard RNA). Eleven (11) rounds of the SELEX experiment werecompleted.

Following completion of 11 rounds of SELEX against FVIIa, cDNAs from therounds 10 and 11 pools were cloned and sequenced. Representativesequences are set forth in Table 4 above. The round 11 pool binds FVIIawith a K_(D) of ˜10 nM under physiologic conditions, which representsa >1000 fold enrichment in binding activity for FVIIa over the course ofthe SELEX process.

The highest affinity aptamers, sequences 10.15 (SEQ ID NO:75) and 11.12(SEQ ID NO:88), both bind FVIIa tightly, with apparent K_(d)'s of ˜5-8nM under physiologic conditions (FIG. 23A). Both aptamers bind thezymogen FVII with similar affinity. Aptamers 10.15 and 11.12 are bothpotent anticoagulants of human plasma in vitro as demonstrated by theirability to prolong Prothrombin Time (PT) clotting assays (FIG. 23B). Thein vitro anticoagulant activity of both aptamers is comparable to theanticoagulant activity of the most potent FVIIa antagonists currentlyavailable in the art. In addition, neither aptamer prolongs the clottingtime of human plasma in Activated Partial Thromboplastin Time (APTT)clotting assays, as expected for specific FVIIa antagonists. Generatingactive truncates of both of these aptamers allows production of theseaptamers by chemical synthesis for in vivo analysis.

Example 12 Inhibition of the Activity of Coagulation Factor Xa

This Example pertains to the isolation of active truncates of aptamersgenerated against coagulation factor Xa (FXa). Shown in FIG. 21 is thepredicted secondary structure of a fully active 36 nucleotide version ofaptamer 11.F7 (SEQ ID NO:148), a sequence related to the aptamer 10.14(SEQ ID NO:73) described above. This aptamer, termed 11.F7^(t), bindsFXa with an apparent K_(d) of 1.5 nM, and exhibits at least severalhundred fold specificity for FXa versus related coagulation factorsVIIa, IXa, XIa, APC and thrombin. This truncate is a potentanticoagulant of human plasma in vitro as demonstrated by its ability toprolong both the PT (FIG. 22A) and APTT (FIG. 22B) clotting times. Basedupon comparison to the in vitro anticoagulant activity of other FXainhibitors described in the art, this aptamer is one of most potent FXaantagonists described. The activity of this aptamer in animal models ofsystemic anticoagulation and thrombosis is also provided, in accordancewith the in vivo experiments disclosed in previous Examples.

Example 13 Modulation of the Activity of Ang1

This Example pertains to an analysis of the modulation of Ang1 by Ang1RNA aptamers. To determine whether ANG9-4 (SEQ ID NO:151) bindinginhibited Ang1 activity, 293 cells expressing human Tie2 were incubatedwith 13 nM Ang1* with or without a molar excess of 9-4 or controlaptamer. ANG9-4 completely abrogated Tie2 autophosphorylation asdetected by Western blotting with an antibody specific for phospho-Tie2(pTie2)(see FIG. 25).

As shown in FIG. 26, cultured human endothelial cells were serum-starvedand treated with TNFa (50 ng/ml) for 3 hours while being incubated with3.5 nM Ang1* and either ANG9-4 or control aptamer. Apoptosis (DNAfragmentation) was measured by Cell Death Detection ELISA kit (RocheMolecular Biochemicals). ANG9-4, but not control aptamer, increasedapoptosis in a dose-dependent manner. Neither 9-4 nor control RNAincreased apoptosis in un-starved, un-treated cells.

Example 14 Modulation of the Activity of Ang2

As shown in FIG. 28, cultured human endothelial cells were serum-starvedand treated with TNFa (50 ng/ml) for 3 hours while being incubated with15 nM Ang2 and either ANG11-1 or control aptamer. Apoptosis (DNAfragmentation) was measured by Cell Death Detection ELISA kit (RocheMolecular Biochemicals). ANG11-1, but not control aptamer, increasedapoptosis to a levels above those seen in the absence of exogenous Ang2,suggesting inhibition of both exogenous and endogenous (autocrine) Ang2,which is known to be released by endothelial cells. Neither ANG11-1 norcontrol RNA increased apoptosis in non-irradiated cells.

As in FIG. 29, the ability of ANG11-1 (SEQ ID NO:168) and a41-nucleotide truncate (ANG11-1.41) (SEQ ID NO:189) to compete forbinding to ANG2 (normalized to binding in the absence of competitor) isplotted as a function of competitor concentration. ANG11-1.41 competesfor ANG2 binding with an affinity (K_(d)˜5 nM) only slightly worse thatof the full-length RNA aptamer (˜1 nM).

REFERENCES

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

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It will be understood that various details of the invention can bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation—the invention being defined by theclaims.

1. An RNA aptamer that binds to the coagulation pathway factorprothrombin or thrombin.
 2. The aptamer of claim 1 wherein the aptameris at least 80% homologous to a nucleotide sequence selected from thegroup comprising SEQ ID NOs: 50-57, or a truncate thereof.
 3. Theaptamer of claim 1 comprising nucleotide sequences selected from thegroup comprising SEQ ID NOs: 50-57, or a truncate thereof.
 4. Theaptamer of claim 1 that binds coagulation pathway factor prothrombin. 5.The aptamer of claim 1 that binds coagulation pathway factor thrombin.6. The aptamer of claim 1, having a dissociation constant of about 100picomolar (pM) to about 10 nanomolar (nM) or less.
 7. The aptamer ofclaim 1, wherein the aptamer comprises at least one 2′-modifiednucleotide.
 8. The aptamer of claim 7 wherein the 2′-modified nucleotideis selected from the group consisting of a 2′-halo-modified nucleotide,a 2′-fluoro-modified nucleotide, and a 2′-O-alkyl-modified nucleotide.9. The aptamer of claim 7 comprising at least one 2′-fluorocitidine orone 2′-fluorouridine.
 10. The aptamer of claim 7, wherein all cytidinesare 2′-deoxy-2′-fluorocytidine, or all uridines are2′-deoxy-2′-fluorouridine.
 11. The aptamer of claim 7 comprising atleast one 2′-O-alkyl-modified nucleotide.
 12. The aptamer of claim 1,wherein the aptamer comprises about 15 to 100 bases.
 13. The aptamer ofclaim 1, wherein the aptamer further comprises a covalently linkedcarrier selected from the group consisting of a soluble polymer, abiodegradable polymer, polyethylene glycol, and cholesterol.
 14. Apharmaceutical composition comprising a therapeutically effective amountof an RNA aptamer which binds to the coagulation pathway factorprothrombin or thrombin.
 15. The composition of claim 14 wherein theaptamer binds coagulation pathway factor prothrombin.
 16. Thecomposition of claim 14 wherein the aptamer binds coagulation pathwayfactor thrombin.
 17. The composition of claim 14 wherein the aptamercomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 50-57.
 18. The composition of claim 14, wherein the aptamercomprises a sequence at least 80% homologous to a nucleotide sequenceselected from SEQ ID NOs; 50-57.
 19. The composition of claim 14 whereinthe aptamer comprises at least one 2′-modified nucleotide.
 20. Thecomposition of claim 19, wherein the aptamer comprises at least onemodified nucleotide selected from the group consisting of a2′-halo-modified nucleotide, a 2′-fluoro-modified nucleotide, and a2′-O-alkyl-modified nucleotide.
 21. The composition of claim 20, whereinthe aptamer comprises cytidines that are all 2′-deoxy-2′-fluorocytidine,or uridines that are all 2′-deoxy-2′-fluorouridine.
 22. The compositionof claim 14, wherein the aptamer further comprises a covalently linkedcarrier selected from the group comprising; a soluble polymer, abiodegradable polymer, polyethylene glycol, or cholesterol.
 23. Thecomposition of claim 14, wherein the composition is in a unit dose. 24.A method of modulating the biological activity of a coagulation pathwayfactor prothrombin or thrombin comprising administering an RNA aptamerthat binds to the coagulation pathway factor prothrombin or thrombin toa host.
 25. The composition of claim 14 wherein the aptamer bindscoagulation pathway factor thrombin